Modulators of resistant androgen receptor

ABSTRACT

The present invention provides compounds useful as modulators, agonists or antagonists of androgen receptor (AR), compositions thereof, and methods of making and using the same. Compounds of the present invention, and pharmaceutically acceptable compositions thereof, are useful in medicine, and particularly for treating any of a variety of diseases, disorders or conditions.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application claims priority to U.S. provisional applicationSer. No. 61/719,117, filed Oct. 26, 2012, the entirety of which isincorporated herein by reference.

BACKGROUND OF THE INVENTION

Prostate cancer is the ninth-most-common cancer in the world, but is thenumber-one non-skin cancer in men from the United States. As of 2011,prostate cancer is the second most frequently diagnosed cancer and thesixth leading cause of cancer death in males worldwide. In 2008, therewere 186,000 new diagnoses and 28,600 deaths attributable to prostatecancer. In India in the 1990s, half of the people with prostate cancerconfined to the prostate died within ten years. The continuing andhighly prevalent problem of prostate cancer highlights the overwhelmingneed for new drugs to treat this condition and its underlying causes.

SUMMARY OF THE INVENTION

It has now been found that compounds of this invention, andpharmaceutically acceptable compositions thereof, are effective asmodulators of androgen receptors (ARs). Such compounds have the generalformula I:

or a pharmaceutically acceptable salt thereof, wherein each variable isas defined and described herein.

Compounds of the present invention, and pharmaceutically acceptablecompositions thereof, are useful in medicine, and particularly fortreating any of a variety of diseases, disorders or conditions. Forexample, provided compounds are useful in treatment of diseases,disorders or conditions associated with ARs, and particularly withdiseases, disorders or conditions associated with androgen-resistant ARsor an AR mutant associated with “castration-resistant” prostate cancer.Such diseases, disorders, or conditions include those described herein.

Compounds provided by this invention are also useful for the study ofandrogen receptors in biological and pathological phenomena; the studyof intracellular signal transduction pathways occurring in reproductiveand other bodily tissues; and the comparative evaluation of new ARmodulators or treatments for AR-related diseases in vitro or in vivo.

In some embodiments, by virtue of their interaction with ARs thecompounds of the present invention are useful as targeting moieties forthe delivery of payloads targeting cancer cells expressing wild type ormutant androgen-resistant ARs or AR mutants associated with“castration-resistant” prostate cancer. Such uses as targeting agentsfor the delivery of payloads include those described herein.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 presents results of an in vitro GFP reporter assay for 1 uM and10 uM concentrations of Enzalutamide (MDV3100) and ARN509.

FIG. 2 presents results of an in vitro GFP reporter assay for 10 uMconcentrations of compounds I-9, rac I-1, and Enzalutamide (MDV3100).

FIG. 3 presents results of an in vitro GFP reporter assay for 10 uMconcentrations of compounds I-10, (±)-I-1, and I-8.

FIG. 4 presents results of an in vitro GFP reporter assay for 10 uMconcentrations of compounds I-5, and (±)-I-1.

FIG. 5 presents results of an in vitro GFP reporter assay for 10 uMconcentrations of compounds I-26, I-11, and Enzalutamide (MDV3100).

FIG. 6 presents results of an in vitro GFP reporter assay for 10 uMconcentrations of compounds I-5, (±)-I-1, and ARN509.

FIG. 7 presents results of an in vitro GFP reporter assay for a 10 uMconcentration of compound I-6.

FIG. 8 presents results of an in vitro GFP reporter assay for 10 uMconcentrations of compounds I-21, I-25, I-22 and I-18.

FIG. 9 presents results of an in vitro GFP reporter assay for 10 uMconcentrations of compounds I-23, I-19, I-24 and I-20.

FIG. 10 presents results of an in vitro GFP reporter assay for 10 uMconcentrations of compounds I-9 and I-14.

FIG. 11 presents results of an in vitro GFP reporter assay for 10 uMconcentrations of compounds (±)-I-1, (±)-I-3 and I-7.

FIG. 12 presents results of an in vitro GFP reporter assay for 10 uMconcentrations of compounds I-11 and I-16.

FIG. 13 presents results of an in vitro GFP reporter assay for 10 uMconcentrations of compounds I-27 and I-26.

FIG. 14 presents results of an in vitro GFP reporter assay for 10 uMconcentrations of compounds I-17 and I-12.

FIG. 15 presents results of an in vitro GFP reporter assay for 10 uMconcentrations of compounds (±)-I-3, I-15, I-14 and I-13.

FIG. 16 presents tabulated results of GFP reporter assay experimentsshowing agonist (+) and antagonist (−) properties of compounds offormula I at both wild-type and F876L mutant AR.

FIG. 17 presents results of an in vitro cell viability assay showing theability of compound I-1 to kill or inhibit the growth of VCaP cellsexpressing either wild type or mutant F876L androgen receptor.

FIG. 18 presents results of an in vitro cell viability assay showing theability of compound I-1 to kill or inhibit the growth of CWR22PC cellsexpressing either wild type or mutant F876L androgen receptor.

FIG. 19 depicts chemical structures and ring naming for AR antagonistcompounds, a crystal structure of bicalutamide bound to AR, and amolecular model of several antagonists in the AR binding pocket.

FIG. 20 depicts Molecular Dynamics Simulations of AR and ARF876LAntagonist Complexes.

FIG. 21 depicts a comparison of AR antagonist models illustratingdisplacements of helices H11 and H12 with a larger ring substituent onthe antagonist.

FIG. 22 depicts a comparison of AR Antagonist Models for AR WT and ARF876L.

FIG. 23 depicts eight overlaid extended molecular dynamics simulationsof AR F876L with MDV3100.

FIG. 24 depicts a zoomed in view of the H11 pocket of FIGS. 22A and 22B.

FIG. 25 depicts results of a luciferase reporter assay showing that I-1effectively competes with dihydrotestosterone (DHT) for AR binding andinduction of AR-regulated luciferase.

FIG. 26 depicts results of a luciferase reporter assay showing that I-1is a more potent agonist for AR F876L than AR WT.

FIG. 27 depicts the ability of I-1 to inhibit AR signaling and inducePARP cleavage in cells expressing both AR WT and AR F876L.

DETAILED DESCRIPTION OF CERTAIN EMBODIMENTS

1. General Description of Compounds of the Invention:

In certain embodiments, the present invention provides modulators,agonists and antagonists of AR. In some embodiments, the presentinvention provides modulators, agonists and antagonists ofandrogen-resistant ARs. In some embodiments, the present inventionprovides modulators, agonists and antagonists of androgen-resistant ARmutants and/or AR mutants that are associated with castration-resistantprostate cancer. In some embodiments, such compounds include those offormula I:

or a pharmaceutically acceptable salt thereof, wherein:

-   X is CH or N; and-   Z is —CH₂— or Ring B; and-   Ring B is an optionally substituted 5-14 membered saturated or    partially unsaturated carbocyclic monocyclic or bicyclic ring,    wherein said ring is spiro-fused at point Z.    2. Compounds and Definitions:

Compounds of this invention include those described generally above, andare further illustrated by the classes, subclasses, and speciesdisclosed herein. As used herein, the following definitions shall applyunless otherwise indicated. For purposes of this invention, the chemicalelements are identified in accordance with the Periodic Table of theElements, CAS version, Handbook of Chemistry and Physics, 75^(th) Ed.Additionally, general principles of organic chemistry are described in“Organic Chemistry”, Thomas Sorrell, University Science Books,Sausalito: 1999, and “March's Advanced Organic Chemistry”, 5^(th) Ed.,Ed.: Smith, M. B. and March, J., John Wiley & Sons, New York: 2001, theentire contents of which are hereby incorporated by reference.

The phrase “spiro fused at point Z”, as used herein, means that when Zis Ring B, said Ring B is spiro-fused to the thiohydantoin ring offormula I in such a manner that both rings share a commontetrasubstituted carbon atom denoted Z in formula I. By way of anon-limiting example, in some embodiments when Ring B is a cyclopentanering, compounds of formula I have the general formula:

Furthermore, as a means for denoting the point of spiro fusion in theembodiments herein, specific examples of Ring B shall be understood tobe fused to the thiohydantoin ring at the carbon atom denoted Z. Thus,as a non-limiting example, when it is desired to communicate that Ring Bis a 4,4-dimethylcyclohexane ring fused to the thiohydantoin ringthrough position 1 of the cyclohexane ring, such a ring can be depictedin the following manner:

Alternatively, the point of attachment on each of the spiro-fused ringscan be denoted by asterisks in the following manner:

The term “aliphatic” or “aliphatic group”, as used herein, means astraight-chain (i.e., unbranched) or branched, substituted orunsubstituted hydrocarbon chain that is completely saturated or thatcontains one or more units of unsaturation, or a monocyclic hydrocarbonor bicyclic hydrocarbon that is completely saturated or that containsone or more units of unsaturation, but which is not aromatic (alsoreferred to herein as “carbocycle,” “cycloaliphatic” or “cycloalkyl”),that has a single point of attachment to the rest of the molecule.Unless otherwise specified, aliphatic groups contain 1-6 aliphaticcarbon atoms. In some embodiments, aliphatic groups contain 1-5aliphatic carbon atoms. In other embodiments, aliphatic groups contain1-4 aliphatic carbon atoms. In still other embodiments, aliphatic groupscontain 1-3 aliphatic carbon atoms, and in yet other embodiments,aliphatic groups contain 1-2 aliphatic carbon atoms. In someembodiments, “cycloaliphatic” (or “carbocycle” or “cycloalkyl”) refersto a monocyclic C₃-C₆ hydrocarbon that is completely saturated or thatcontains one or more units of unsaturation, but which is not aromatic,that has a single point of attachment to the rest of the molecule.Suitable aliphatic groups include, but are not limited to, linear orbranched, substituted or unsubstituted alkyl, alkenyl, alkynyl groupsand hybrids thereof such as (cycloalkyl)alkyl, (cycloalkenyl)alkyl or(cycloalkyl)alkenyl.

The term “lower alkyl” refers to a C₁₋₄ straight or branched alkylgroup. Exemplary lower alkyl groups are methyl, ethyl, propyl,isopropyl, butyl, isobutyl, and tert-butyl.

The term “lower haloalkyl” refers to a C₁₋₄ straight or branched alkylgroup that is substituted with one or more halogen atoms.

As used herein, the term “androgen” is used herein to refer to agentsandrogenic activity. Androgenic activity may be determined orcharacterized in any of a variety of ways, including in any of a varietyof biological activity assays (e.g., in vitro or in vivo assays, forexample utilizing animals and/or animal tissues) in which the agent isobserved to have one or more activities similar or comparable to that ofa known androgen assessed under comparable conditions (whethersimultaneously or otherwise). In some embodiments, androgenic activityis or comprises transcriptional regulation (e.g., activation) of anandrogen-responsive target gene. In some embodiments, androgenicactivity is or comprises stimulation of prostate growth in rodents.Exemplary know androgens include, for example, androstanedione,androstenediol, androstenedione, androsterone, dehydroepiandrosterone,dehydroepiandrosterone sulfate, dihydrotestosterone (DHT), andtestosterone.

As used herein, the term “antiandrogen” refers to any agent thatinhibits biological activity of androgens. In some embodiments,antiandrogens inhibit biological activity of an AR. In some embodimentsantiandrogens inhibit biological activity of a wild type AR. In someembodiments, antiandrogens inhibit biological activity of one or more ARincluded in Table A. In some embodiments, antiandrogens compete with oneor more androgens for binding to an AR. In some embodiments,antiandrogens compete with one or more androgens for binding to a wildtype AR. In some embodiments, antiandrgens compete with one or moreandrogens for binding to an AR included in Table A. In some embodiments,antiandrogens comprise 3,3′-diindolylmethane (DIM), ARN-509,bexlosteride, bicalutamide, N-butylbenzene-sulfonamide (NBBS),dutasteride, epristeride, enzalutamide, finasteride, flutamide,izonsteride, ketoconazole, N-butylbenzene-sulfonamide, nilutamide,megestrol, steroidal antiandrogens, and/or turosteride.

As used herein, the term “associated with” means correlated with orstatistically likely to occur or appear together with another state, orcondition. In some embodiments, the term “associated with” refers tostatistically non-random or correlated events. In some embodiments, theterm “associated with” refers to a physical association inthree-dimensional space. In some such embodiments, such physicalassociation is mediated by one or more covalent or non-covalent (e.g.,hydrogen bonds, hydrophobic interactions, van der Waals interactions,electrostatic forces, magnetic forces, pi-pi interactions, sigma-piinteractions, etc.).

As used herein, the terms “correlates” or “correlated”, as used herein,has its ordinary meaning of “showing a correlation with”. Those ofordinary skill in the art will appreciate that two features, items orvalues show a correlation with one another if they show a tendency toappear and/or to vary, together. In some embodiments, a correlation isstatistically significant when its p-value is less than 0.05; in someembodiments, a correlation is statistically significant when its p-valueis less than 0.01. In some embodiments, correlation is assessed byregression analysis. In some embodiments, a correlation is a correlationcoefficient.

As used herein, the term “corresponding to” is often used to designatethe position/identity of a particular residue within a polymeric agent(e.g., within a nucleic acid or polypeptide). Those of ordinary skillwill appreciate that, for purposes of simplicity, a canonical numberingsystem (based on a reference polymer) is often utilized herein in orderto facilitate comparison of polymer sequences. Those of ordinary skillin the art understand how to align polymer sequences in order todetermine which residues “correspond” to particular positions in areference polymer. For example, those skilled in the art appreciate thata particular residue in a polypeptide of interest may “correspond to” aresidue at a certain position in a reference polypeptide even if it isnot found at the same position (relative to a terminus of thepolypeptide) in the polypeptide of interest, so long as its context inthe polypeptide of interest is sufficiently similar to that of theresidue in the polypeptide of interest that it would be recognized byone skilled in the art as “corresponding to” that reference residue.

As used herein, a “detection moiety” in the context of providedmultifunctional agents refers to a molecular structure or module thatallows visualization/imaging, measurements (localization,quantification, etc.) and/or monitoring of an agent in vitro and/or invivo using one or more detection techniques including but not limited tospectroscopic, photochemical, biochemical, immunochemical, electrical,optical, chemical or other means.

The term “heteroatom” means one or more of oxygen, sulfur, nitrogen,phosphorus, or silicon (including, any oxidized form of nitrogen,sulfur, phosphorus, or silicon; the quaternized form of any basicnitrogen or; a substitutable nitrogen of a heterocyclic ring, forexample N (as in 3,4-dihydro-2H-pyrrolyl), NH (as in pyrrolidinyl) orNR⁺ (as in N-substituted pyrrolidinyl)).

As used herein, the terms “improve,” “increase” or “reduce,” orgrammatical equivalents, indicate a change in a value relative to acomparable baseline or reference measurement. In some embodiments, acomparable baseline or reference measurement is a measurement taken inthe same system (e.g., of the same individual) prior to initiation of anevent of interest (e.g., of therapy). In some embodiments, a comparablebaseline or reference measurement is one taken in a different system(e.g., a different individual or cell) under otherwise identicalconditions (e.g., in a normal cell or individual as compared with onesuffering from or susceptible to a particular disease, disorder orcondition, for example due to presence of a particular geneticmutation).

As used herein, the term “in vitro” refers to events that occur in anartificial environment, e.g., in a test tube or reaction vessel, in cellculture, etc., rather than within a multi-cellular organism.

As used herein, the term “in vivo” refers to events that occur within amulti-cellular organism, such as a human and a non-human animal. In thecontext of cell-based systems, the term may be used to refer to eventsthat occur within a living cell (as opposed to, for example, in vitrosystems).

As used herein, the term “mutant” refers to an altered (as compared witha reference) nucleic acid or polypeptide, or to a cell or organismcontaining or expressing such an altered nucleic acid or polypeptide.

The term “polypeptide” or “peptide”, as used herein, generally has itsart-recognized meaning of a polymer of at least three amino acids. Thoseof ordinary skill in the art will appreciate that the term “polypeptide”is intended to be sufficiently general as to encompass not onlypolypeptides having the a complete sequence recited herein, but also toencompass polypeptides that represent functional fragments (i.e.,fragments retaining at least one activity) of such completepolypeptides. Moreover, those of ordinary skill in the art understandthat protein sequences generally tolerate some substitution withoutdestroying activity. Thus, any polypeptide that retains activity andshares at least about 30-40% overall sequence identity, often greaterthan about 50%, 60%, 70%, or 80%, and further usually including at leastone region of much higher identity, often greater than 90% or even 95%,96%, 97%, 98%, or 99% in one or more highly conserved regions, usuallyencompassing at least 3-4 and often up to 20 or more amino acids, withanother polypeptide of the same class, is encompassed within therelevant term “polypeptide” as used herein.

The term “protein” as used herein refers to one or more polypeptidesthat function as a discrete unit. If a single polypeptide is thediscrete functioning unit and does not require permanent or temporaryphysical association with other polypeptides in order to form thediscrete functioning unit, the terms “polypeptide” and “protein” may beused interchangeably. If the discrete functional unit is comprised ofmore than one polypeptide that physically associate with one another,the term “protein” may be used to refers to the multiple polypeptidesthat are physically associated coupled and function together as thediscrete unit.

As will be understood from context, a reference sequence, sample,population, agent or individual is one that is sufficiently similar to aparticular sequence, sample, population, agent or individual of interestto permit a relevant comparison (i.e., to be comparable). In someembodiments, information about a reference sample is obtainedsimultaneously with information about a particular sample. In someembodiments, information about a reference sample is historical. In someembodiments, information about a reference sample is stored for examplein a computer-readable medium. In some embodiments, comparison of aparticular sample of interest with a reference sample establishesidentity with, similarity to, or difference of a particular sample ofinterest relative to a reference.

As used herein, the term “sample” typically refers to a biologicalsample obtained or derived from a source of interest, as describedherein. In some embodiments, a source of interest comprises an organism,such as an animal or human. In some embodiments, a biological sample isor comprises biological tissue or fluid. In some embodiments, abiological sample may be or comprise bone marrow; blood; blood cells;ascites; tissue or fine needle biopsy samples; cell-containing bodyfluids; free floating nucleic acids; sputum; saliva; urine;cerebrospinal fluid, peritoneal fluid; pleural fluid; feces; lymph;gynecological fluids; skin swabs; vaginal swabs; oral swabs; nasalswabs; washings or lavages such as a ductal lavages or broncheoalveolarlavages; aspirates; scrapings; bone marrow specimens; tissue biopsyspecimens; surgical specimens; feces, other body fluids, secretions,and/or excretions; and/or cells therefrom, etc. In some embodiments, abiological sample is or comprises cells obtained from an individual. Insome embodiments, obtained cells are or include cells from an individualfrom whom the sample is obtained. In some embodiments, a sample is a“primary sample” obtained directly from a source of interest by anyappropriate means. For example, in some embodiments, a primarybiological sample is obtained by methods selected from the groupconsisting of biopsy (e.g., fine needle aspiration or tissue biopsy),surgery, collection of body fluid (e.g., blood, lymph, feces etc.), etc.In some embodiments, as will be clear from context, the term “sample”refers to a preparation that is obtained by processing (e.g., byremoving one or more components of and/or by adding one or more agentsto) a primary sample. For example, filtering using a semi-permeablemembrane. Such a “processed sample” may comprise, for example nucleicacids or proteins extracted from a sample or obtained by subjecting aprimary sample to techniques such as amplification or reversetranscription of mRNA, isolation and/or purification of certaincomponents, etc.

As used herein, a “targeting moiety” in the context of providedmultifunctional agents refers to a molecular structure or module thataffects or controls the site of action by specifically interacting with,or has affinity for, a target of interest. In some embodiments, atargeting moiety useful for the present invention is a compound offormula I.

As used herein, a “therapeutic moiety” in the context of providedmultifunctional agents refers to a molecular structure or module thatconfers a therapeutic effect. In some embodiments, therapeutic effectsconferred by a therapeutic moiety of a multifunctional agent of thepresent invention include anti-cancer effects. Accordingly, atherapeutic moiety may be an anti-cancer agent (e.g., chemotherapeuticagent). In some embodiments, anti-cancer agents useful for the presentinvention are agents that inhibit tumor growth, agents that inhibitproliferation of cancer cells, agents that preferentially kill cancercells, agents that inhibit angiogenesis, etc. In some embodiments, suchagents are small molecules. Small molecules include, without limitation,small chemical-based entities, such as chemotoxins and cytostatic drugs,which may be referred to as “SCEs.” Typically, SCEs are non-peptide,non-nucleic acid molecules. In those embodiments that include both atherapeutic entity and a diagnostic entity, the therapeutic entity andthe target entity are not the same entity.

As used herein, the term “therapeutically effective amount” refers to anamount of an agent which confers a therapeutic effect on a treatedsubject, at a reasonable benefit/risk ratio applicable to any medicaltreatment. A therapeutic effect may be objective (i.e., measurable bysome test or marker) or subjective (i.e., subject gives an indication ofor feels an effect). In particular, a “therapeutically effective amount”refers to an amount of a therapeutic agent effective to treat,ameliorate, or prevent a desired disease or condition, or to exhibit adetectable therapeutic or preventative effect, such as by amelioratingsymptoms associated with a disease, preventing or delaying onset of adisease, and/or also lessening severity or frequency of symptoms of adisease. A therapeutically effective amount is commonly administered ina dosing regimen that may comprise multiple unit doses. For anyparticular therapeutic agent, a therapeutically effective amount (and/oran appropriate unit dose within an effective dosing regimen) may vary,for example, depending on route of administration, on combination withother agents. Also, a specific therapeutically effective amount (and/orunit dose) for any particular patient may depend upon a variety offactors including what disorder is being treated; disorder severity;activity of specific agents employed; specific composition employed;age, body weight, general health, and diet of a patient; time ofadministration, route of administration; treatment duration; and likefactors as is well known in the medical arts.

As used herein, the term “treat,” “treatment,” or “treating” refers toany method used to partially or completely alleviate, ameliorate,relieve, inhibit, prevent, delay onset of, reduce severity of and/orreduce incidence of one or more symptoms or features of a particulardisease, disorder, and/or condition. Treatment may be administered to asubject who does not exhibit signs of a disease and/or exhibits onlyearly signs of the disease for the purpose of decreasing the risk ofdeveloping pathology associated with the disease.

The term “unsaturated,” as used herein, means that a moiety has one ormore units of unsaturation.

As used herein, the term “wild-type” refers to a typical or common formexisting in nature; in some embodiments it is the most common form.

As used herein, the term “bivalent C₁₋₈ (or C₁₋₆) saturated orunsaturated, straight or branched, hydrocarbon chain”, refers tobivalent alkylene, alkenylene, and alkynylene chains that are straightor branched as defined herein.

The term “alkylene” refers to a bivalent alkyl group. An “alkylenechain” is a polymethylene group, i.e., —(CH₂)_(n)—, wherein n is apositive integer, preferably from 1 to 6, from 1 to 4, from 1 to 3, from1 to 2, or from 2 to 3. A substituted alkylene chain is a polymethylenegroup in which one or more methylene hydrogen atoms are replaced with asubstituent. Suitable substituents include those described below for asubstituted aliphatic group.

The term “alkenylene” refers to a bivalent alkenyl group. A substitutedalkenylene chain is a polymethylene group containing at least one doublebond in which one or more hydrogen atoms are replaced with asubstituent. Suitable substituents include those described below for asubstituted aliphatic group.

As used herein, the term “cyclopropylenyl” refers to a bivalentcyclopropyl group of the following structure:

As used herein, the term “cyclobutylenyl” refers to a bivalentcyclobutyl group of the following structure:

As used herein, the term “oxetanyl” refers to a bivalent oxetanyl groupof the following structure:

The term “halogen” means F, Cl, Br, or I.

The term “aryl” used alone or as part of a larger moiety as in“aralkyl,” “aralkoxy,” or “aryloxyalkyl,” refers to monocyclic orbicyclic ring systems having a total of five to fourteen ring members,wherein at least one ring in the system is aromatic and wherein eachring in the system contains 3 to 7 ring members. The term “aryl” may beused interchangeably with the term “aryl ring.”

The term “aryl” used alone or as part of a larger moiety as in“aralkyl,” “aralkoxy,” or “aryloxyalkyl,” refers to monocyclic andbicyclic ring systems having a total of five to 10 ring members, whereinat least one ring in the system is aromatic and wherein each ring in thesystem contains three to seven ring members. The term “aryl” may be usedinterchangeably with the term “aryl ring”. In certain embodiments of thepresent invention, “aryl” refers to an aromatic ring system whichincludes, but not limited to, phenyl, biphenyl, naphthyl, anthracyl andthe like, which may bear one or more substituents. Also included withinthe scope of the term “aryl,” as it is used herein, is a group in whichan aromatic ring is fused to one or more non-aromatic rings, such asindanyl, phthalimidyl, naphthimidyl, phenanthridinyl, ortetrahydronaphthyl, and the like.

The terms “heteroaryl” and “heteroar-,” used alone or as part of alarger moiety, e.g., “heteroaralkyl,” or “heteroaralkoxy,” refer togroups having 5 to 10 ring atoms, preferably 5, 6, or 9 ring atoms;having 6, 10, or 14 π electrons shared in a cyclic array; and having, inaddition to carbon atoms, from one to five heteroatoms. The term“heteroatom” refers to nitrogen, oxygen, or sulfur, and includes anyoxidized form of nitrogen or sulfur, and any quaternized form of a basicnitrogen. Heteroaryl groups include, without limitation, thienyl,furanyl, pyrrolyl, imidazolyl, pyrazolyl, triazolyl, tetrazolyl,oxazolyl, isoxazolyl, oxadiazolyl, thiazolyl, isothiazolyl,thiadiazolyl, pyridyl, pyridazinyl, pyrimidinyl, pyrazinyl, indolizinyl,purinyl, naphthyridinyl, and pteridinyl. The terms “heteroaryl” and“heteroar-”, as used herein, also include groups in which aheteroaromatic ring is fused to one or more aryl, cycloaliphatic, orheterocyclyl rings, where the radical or point of attachment is on theheteroaromatic ring. Nonlimiting examples include indolyl, isoindolyl,benzothienyl, benzofuranyl, dibenzofuranyl, indazolyl, benzimidazolyl,benzthiazolyl, quinolyl, isoquinolyl, cinnolinyl, phthalazinyl,quinazolinyl, quinoxalinyl, 4H-quinolizinyl, carbazolyl, acridinyl,phenazinyl, phenothiazinyl, phenoxazinyl, tetrahydroquinolinyl,tetrahydroisoquinolinyl, and pyrido[2,3-b]-1,4-oxazin-3(4H)-one. Aheteroaryl group may be mono- or bicyclic. The term “heteroaryl” may beused interchangeably with the terms “heteroaryl ring,” “heteroarylgroup,” or “heteroaromatic,” any of which terms include rings that areoptionally substituted. The term “heteroaralkyl” refers to an alkylgroup substituted by a heteroaryl, wherein the alkyl and heteroarylportions independently are optionally substituted.

As used herein, the terms “heterocycle,” “heterocyclyl,” “heterocyclicradical,” and “heterocyclic ring” are used interchangeably and refer toa stable 5- to 7-membered monocyclic or 7-10-membered bicyclicheterocyclic moiety that is either saturated or partially unsaturated,and having, in addition to carbon atoms, one or more, preferably one tofour, heteroatoms, as defined above. When used in reference to a ringatom of a heterocycle, the term “nitrogen” includes a substitutednitrogen. As an example, in a saturated or partially unsaturated ringhaving 0-3 heteroatoms selected from oxygen, sulfur or nitrogen, thenitrogen may be N (as in 3,4-dihydro-2H-pyrrolyl), NH (as inpyrrolidinyl), or ⁺NR (as in N-substituted pyrrolidinyl).

A heterocyclic ring can be attached to its pendant group at anyheteroatom or carbon atom that results in a stable structure and any ofthe ring atoms can be optionally substituted. Examples of such saturatedor partially unsaturated heterocyclic radicals include, withoutlimitation, tetrahydrofuranyl, tetrahydrothiophenyl pyrrolidinyl,piperidinyl, pyrrolinyl, tetrahydroquinolinyl, tetrahydroisoquinolinyl,decahydroquinolinyl, oxazolidinyl, piperazinyl, dioxanyl, dioxolanyl,diazepinyl, oxazepinyl, thiazepinyl, morpholinyl, and quinuclidinyl. Theterms “heterocycle,” “heterocyclyl,” “heterocyclyl ring,” “heterocyclicgroup,” “heterocyclic moiety,” and “heterocyclic radical,” are usedinterchangeably herein, and also include groups in which a heterocyclylring is fused to one or more aryl, heteroaryl, or cycloaliphatic rings,such as indolinyl, 3H-indolyl, chromanyl, phenanthridinyl, ortetrahydroquinolinyl, where the radical or point of attachment is on theheterocyclyl ring. A heterocyclyl group may be mono- or bicyclic. Theterm “heterocyclylalkyl” refers to an alkyl group substituted by aheterocyclyl, wherein the alkyl and heterocyclyl portions independentlyare optionally substituted.

As used herein, the term “partially unsaturated” refers to a ring moietythat includes at least one double or triple bond. The term “partiallyunsaturated” is intended to encompass rings having multiple sites ofunsaturation, but is not intended to include aryl or heteroarylmoieties, as herein defined.

As described herein, compounds of the invention may contain “optionallysubstituted” moieties. In general, the term “substituted,” whetherpreceded by the term “optionally” or not, means that one or morehydrogens of the designated moiety are replaced with a suitablesubstituent. Unless otherwise indicated, an “optionally substituted”group may have a suitable substituent at each substitutable position ofthe group, and when more than one position in any given structure may besubstituted with more than one substituent selected from a specifiedgroup, the substituent may be either the same or different at everyposition. Combinations of substituents envisioned by this invention arepreferably those that result in the formation of stable or chemicallyfeasible compounds. The term “stable,” as used herein, refers tocompounds that are not substantially altered when subjected toconditions to allow for their production, detection, and, in certainembodiments, their recovery, purification, and use for one or more ofthe purposes disclosed herein.

Suitable monovalent substituents on a substitutable carbon atom of an“optionally substituted” group are independently halogen;—(CH₂)₀₋₄R^(∘); —(CH₂)₀₋₄OR^(∘); —O(CH₂)₀₋₄R^(∘), —O—(CH₂)₀₋₄C(O)OR^(∘);—(CH₂)₀₋₄CH(OR^(∘))₂; —(CH₂)₀₋₄SR^(∘); —(CH₂)₀₋₄Ph, which may besubstituted with R^(∘); —(CH₂)₀₋₄O(CH₂)₀₋₁Ph which may be substitutedwith R^(∘); —CH═CHPh, which may be substituted with R^(∘);—(CH₂)₀₋₄O(CH₂)₀₋₁-pyridyl which may be substituted with R^(∘); —NO₂;—CN; —N₃; —(CH₂)₀₋₄N(R^(∘))₂; —(CH₂)₀₋₄N(R^(∘))C(O)R^(∘);—N(R^(∘))C(S)R^(∘); —(CH₂)₀₋₄N(R^(∘))C(O)NR^(∘) ₂; —N(R^(∘))C(S)NR^(∘)₂; —(CH₂)₀₋₄N(R^(∘))C(O)OR^(∘); —N(R^(∘))N(R^(∘))C(O)R^(∘);—N(R^(∘))N(R^(∘))C(O)NR^(∘) ₂; —N(R^(∘))N(R^(∘))C(O)OR^(∘);—(CH₂)₀₋₄C(O)R^(∘); —C(S)R^(∘); —(CH₂)₀₋₄C(O)OR^(∘);—(CH₂)₀₋₄C(O)SR^(∘); —(CH₂)₀₋₄C(O)OSiR^(∘) ₃; —(CH₂)₀₋₄OC(O)R^(∘);—OC(O)(CH₂)₀₋₄SR—, SC(S)SR^(∘); —(CH₂)₀₋₄SC(O)R^(∘); —(CH₂)₀₋₄C(O)NR^(∘)₂; —C(S)NR^(∘) ₂; —C(S)SR^(∘); —SC(S)SR^(∘), —(CH₂)₀₋₄OC(O)NR^(∘) ₂;—C(O)N(OR^(∘))R^(∘); —C(O)C(O)R^(∘); —C(O)CH₂C(O)R^(∘);—C(NOR^(∘))R^(∘); —(CH₂)₀₋₄SSR^(∘); —(CH₂)₀₋₄S(O)₂R^(∘); —(CH₂)₀₋₄S(O)₂OR^(∘); —(CH₂)₀₋₄OS(O)₂R^(∘); —S(O)₂NR^(∘) ₂; —(CH₂)₀₋₄ S(O)R^(∘);—N(R^(∘))S(O)₂NR^(∘) ₂; —N(R^(∘))S(O)₂R^(∘); —N(OR^(∘))R^(∘);—C(NH)NR^(∘) ₂; —P(O)₂R^(∘); —P(O)R^(∘) ₂; —OP(O)R^(∘) ₂;—OP(O)(OR^(∘))₂; SiR^(∘) ₃; —(C₁₋₄ straight orbranched)alkylene)O—N(R^(∘) ₂; or —(C₁₋₄ straight orbranched)alkylene)C(O)O—N(R^(∘))₂, wherein each R^(∘) may be substitutedas defined below and is independently hydrogen, C₁₋₆ aliphatic, —CH₂Ph,—O(CH₂)₀₋₁Ph, —CH₂-(5-6 membered heteroaryl ring), or a 5-6-memberedsaturated, partially unsaturated, or aryl ring having 0-4 heteroatomsindependently selected from nitrogen, oxygen, or sulfur, or,notwithstanding the definition above, two independent occurrences ofR^(∘), taken together with their intervening atom(s), form a3-12-membered saturated, partially unsaturated, or aryl mono- orbicyclic ring having 0-4 heteroatoms independently selected fromnitrogen, oxygen, or sulfur, which may be substituted as defined below.

Suitable monovalent substituents on R^(∘) (or the ring formed by takingtwo independent occurrences of R^(∘) together with their interveningatoms), are independently halogen, —(CH₂)₀₋₂R^(●), -(haloR^(●)),—(CH₂)₀₋₂OH, —(CH₂)₀₋₂OR^(●), —(CH₂)₀₋₂CH(OR^(●))₂; —O(halo R^(●)), —CN,—N₃, —(CH₂)₀₋₂C(O)R^(●), —(CH₂)₀₋₂C(O)OH, —(CH₂)₀₋₂C(O)OR^(●),—(CH₂)₀₋₂SR^(●), —(CH₂)₀₋₂SH, —(CH₂)₀₋₂NH₂, —(CH₂)₀₋₂NHR^(●),—(CH₂)₀₋₂NR^(●) ₂, —NO₂, —SiR^(●) ₃, —OSiR^(●) ₃, —C(O)SR^(●), —(C₁₋₄straight or branched alkylene)C(O)OR^(●), or —SSR^(●) wherein each R^(●)is unsubstituted or where preceded by “halo” is substituted only withone or more halogens, and is independently selected from C₁ aliphatic,—CH₂Ph, —O(CH₂)₀₋₁Ph, or a 5-6-membered saturated, partiallyunsaturated, or aryl ring having 0-4 heteroatoms independently selectedfrom nitrogen, oxygen, or sulfur. Suitable divalent substituents on asaturated carbon atom of R^(∘) include ═O and ═S.

Suitable divalent substituents on a saturated carbon atom of an“optionally substituted” group include the following: ═O, ═S, ═NNR*₂,═NNHC(O)R*, ═NNHC(O)OR*, ═NNHS(O)₂R*, ═NR*, ═NOR*, —O(C(R*₂))₂₋₃O—, or—S(C(R*₂))₂₋₃S—, wherein each independent occurrence of R* is selectedfrom hydrogen, C₁₋₆ aliphatic which may be substituted as defined below,or an unsubstituted 5-6-membered saturated, partially unsaturated, oraryl ring having 0-4 heteroatoms independently selected from nitrogen,oxygen, or sulfur. Suitable divalent substituents that are bound tovicinal substitutable carbons of an “optionally substituted” groupinclude: —O(CR*₂)₂₋₃O—, wherein each independent occurrence of R* isselected from hydrogen, C₁₋₆ aliphatic which may be substituted asdefined below, or an unsubstituted 5-6-membered saturated, partiallyunsaturated, or aryl ring having 0-4 heteroatoms independently selectedfrom nitrogen, oxygen, or sulfur.

Suitable substituents on the aliphatic group of R* include halogen,—R^(●), -(haloR^(●)), —OH, —OR^(●), —O(haloR^(●)), —CN, —C(O)OH,—C(O)OR^(●), —NH₂, —NHR^(●), —NR^(●) ₂, or —NO₂, wherein each R^(●) isunsubstituted or where preceded by “halo” is substituted only with oneor more halogens, and is independently C₁₋₄ aliphatic, —CH₂Ph,—O(CH₂)₀₋₁Ph, or a 5-6-membered saturated, partially unsaturated, oraryl ring having 0-4 heteroatoms independently selected from nitrogen,oxygen, or sulfur.

Suitable substituents on a substitutable nitrogen of an “optionallysubstituted” group include —R^(†), —NR^(†) ₂, —C(O)R^(†), —C(O)OR^(†),—C(O)C(O)R^(†), —C(O)CH₂C(O)R^(†), —S(O)₂R^(†), —S(O)₂NR^(†) ₂,—C(S)NR^(†) ₂, —C(NH)NR^(†) ₂, or —N(R^(†))S(O)₂R^(†); wherein eachR^(†) is independently hydrogen, C₁₋₆ aliphatic which may be substitutedas defined below, unsubstituted —OPh, or an unsubstituted 5-6-memberedsaturated, partially unsaturated, or aryl ring having 0-4 heteroatomsindependently selected from nitrogen, oxygen, or sulfur, or,notwithstanding the definition above, two independent occurrences ofR^(†), taken together with their intervening atom(s) form anunsubstituted 3-12-membered saturated, partially unsaturated, or arylmono- or bicyclic ring having 0-4 heteroatoms independently selectedfrom nitrogen, oxygen, or sulfur.

Suitable substituents on the aliphatic group of R^(†) are independentlyhalogen, —R^(●), -(haloR^(●)), —OH, —OR^(●), —O(haloR^(●)), —CN,—C(O)OH, —C(O)OR^(●), —NH₂, —NHR^(●), —NR^(●) ₂, or —NO₂, wherein eachR^(●) is unsubstituted or where preceded by “halo” is substituted onlywith one or more halogens, and is independently C₁₋₄ aliphatic, —CH₂Ph,—O(CH₂)₀₋₁Ph, or a 5-6-membered saturated, partially unsaturated, oraryl ring having 0-4 heteroatoms independently selected from nitrogen,oxygen, or sulfur.

As used herein, the term “pharmaceutically acceptable salt” refers tothose salts which are, within the scope of sound medical judgment,suitable for use in contact with the tissues of humans and lower animalswithout undue toxicity, irritation, allergic response and the like, andare commensurate with a reasonable benefit/risk ratio. Pharmaceuticallyacceptable salts are well known in the art. For example, S. M. Berge etal., describe pharmaceutically acceptable salts in detail in J.Pharmaceutical Sciences, 1977, 66, 1-19, incorporated herein byreference. Pharmaceutically acceptable salts of the compounds of thisinvention include those derived from suitable inorganic and organicacids and bases. Examples of pharmaceutically acceptable, nontoxic acidaddition salts are salts of an amino group formed with inorganic acidssuch as hydrochloric acid, hydrobromic acid, phosphoric acid, sulfuricacid and perchloric acid or with organic acids such as acetic acid,oxalic acid, maleic acid, tartaric acid, citric acid, succinic acid ormalonic acid or by using other methods used in the art such as ionexchange. Other pharmaceutically acceptable salts include adipate,alginate, ascorbate, aspartate, benzenesulfonate, benzoate, bisulfate,borate, butyrate, camphorate, camphorsulfonate, citrate,cyclopentanepropionate, digluconate, dodecylsulfate, ethanesulfonate,formate, fumarate, glucoheptonate, glycerophosphate, gluconate,hemisulfate, heptanoate, hexanoate, hydroiodide,2-hydroxy-ethanesulfonate, lactobionate, lactate, laurate, laurylsulfate, malate, maleate, malonate, methanesulfonate,2-naphthalenesulfonate, nicotinate, nitrate, oleate, oxalate, palmitate,pamoate, pectinate, persulfate, 3-phenylpropionate, phosphate, pivalate,propionate, stearate, succinate, sulfate, tartrate, thiocyanate,p-toluenesulfonate, undecanoate, valerate salts, and the like.

Salts derived from appropriate bases include alkali metal, alkalineearth metal, ammonium and N⁺(C₁₋₄alkyl)₄ salts. Representative alkali oralkaline earth metal salts include sodium, lithium, potassium, calcium,magnesium, and the like. Further pharmaceutically acceptable saltsinclude, when appropriate, nontoxic ammonium, quaternary ammonium, andamine cations formed using counterions such as halide, hydroxide,carboxylate, sulfate, phosphate, nitrate, loweralkyl sulfonate and arylsulfonate.

Those skilled in the art will appreciate that some structures providedherein represent compounds that can exist in a plurality of isomeric(e.g., enantiomeric, diastereomeric, and geometric (or conformational))forms; for example, the R and S configurations for each asymmetriccenter, Z and E double bond isomers, and Z and E conformational isomers.Single such isomers or forms (e.g. stereochemical isomers [e.g.,enantiomeric, diastereomeric, etc.] and/or geometric (or conformational)isomers), as well as combinations or mixtures of such forms are withinthe scope of the invention. Similarly, all tautomeric forms of thecompounds of the invention are within the scope of the invention.Additionally, those skilled in the art will appreciate that analogs ofcompounds having depicted structures that vary from the depictedcompound only in the presence of one or more isotopically enriched atomsmay readily be prepared; such “isotopic isomers” of provided compoundsare also within the scope of the present invention, and may be providedindividually or together with one or more other forms of the compound.For example, compounds having depicted structures wherein one or morehydrogen atoms has/have been replaced by deuterium and/or tritium, oneor more carbon atoms has/have been replaced by a ¹³C- or ¹⁴C-enrichedcarbon are within the scope of this invention. Such isotopic isomers areuseful, for example, as analytical tools, as probes in biologicalassays, or as therapeutic agents in accordance with the presentinvention.

3. Description of Exemplary Embodiments:

Androgen Receptor

The androgen receptor (AR), located on Xql 1-12, is a 110 kD nuclearreceptor that, upon activation by androgens, mediates transcription oftarget genes that modulate growth and differentiation of prostateepithelial cells. Similar to other steroid receptors, unbound AR ismainly located in cytoplasm and associated with a complex of heat shockproteins (HSPs) through interactions with its ligand-binding domain.Upon agonist binding, AR undergoes a series of conformational changes:heat shock proteins dissociate from AR, and transformed AR undergoesdimerization, phosphorylation, and nuclear translocation, which ismediated by its nuclear localization signal. Translocated receptors thenbinds to androgen response elements (ARE), which are characterized by asix-nucleotide half-site consensus sequence 5′-TGTTCT-3′ spaced by threerandom nucleotides and are located in promoter or enhancer regions of ARgene targets. Recruitment of other transcription co-regulators(including co-activators and co-repressors) and transcriptionalmachinery further ensures transactivation of AR-regulated geneexpression. All of these processes are initiated by the ligand-inducedconformational changes in the ligand-binding domain.

As used herein, “Androgen-dependent disorder” refers to any disorderthat can benefit from a decrease in androgen stimulation and includespathological conditions that depend on androgen stimulation. An“androgen-dependent disorder” can result from an excessive accumulationof testosterone or other androgenic hormone, increased sensitivity ofandrogen receptors to androgen, or an increase in androgen-stimulatedtranscription. Examples of “androgen-dependent disorders” includeprostate cancer and skin disorders such as, for example, acne,seborrhea, hirsutism, alopecia, or hidradenitis suppurativa.

Prostate Cancer

Prostate cancer is the second most common cause of cancer death in menin the US, and approximately one in every six American men will bediagnosed with the disease during his lifetime. Treatment aimed ateradicating the tumor is unsuccessful in 30% of men, who developrecurrent disease that is usually manifest first as a rise in plasmaprostate-specific antigen (PSA) followed by spread to distant sites.

AR signaling is crucial for development and maintenance of malereproductive organs including prostate glands, as genetic malesharboring loss of function AR mutations and mice engineered with ARdefects do not develop prostates or prostate cancer. This dependence ofprostate cells on AR signaling continues even upon neoplastictransformation.

Given that prostate cancer cells depend on AR for their proliferationand survival, these men are treated with agents that block production oftestosterone (e.g. GnRH agonists), alone or in combination withantiandrogens, which antagonize effects of any residual testosterone.This approach is effective as evidenced by a drop in PSA and regressionof any visible tumor.

Castration Resistant Prostate Cancer

This hormone-refractory state to which most patients eventuallyprogresses in the presence of continued androgen ablation oranti-androgen therapy is known as “castration resistant” prostate cancer(CRPC).

Compelling data demonstrate that AR is expressed in most prostate cancercells and overexpression of AR is necessary and sufficient forandrogen-independent growth of prostate cancer cells. Failure inhormonal therapy, resulting from development of androgen-independentgrowth, is an obstacle for successful management of advanced prostatecancer. Instances of antiandrogen withdrawal syndrome have also beenreported after prolonged treatment with antiandrogens. Antiandrogenwithdrawal syndrome is commonly observed clinically and is defined interms of tumor regression or symptomatic relief observed upon cessationof antiandrogen therapy. AR mutations that result in receptorpromiscuity and the ability of these antiandrogens to exhibit agonistactivity might at least partially account for this phenomenon. Forexample, hydroxyflutamide and bicalutamide act as AR agonists in T8787A,W741L and W741C AR mutants, respectively.

Treatment options for CPRC are an unmet need. Until recently, docetaxelwas the only agent shown to prolong survival. More recently, four newertreatments have come onto the market, including sipuleucel-T, animmunotherapeutic agent; cabazitaxel, a novel microtubule inhibitor;abiraterone acetate, a new androgen biosynthesis inhibitor; anddenosumab. Interestingly, while a small minority of CRPC does bypass therequirement for AR signaling, the vast majority of CRPC, thoughfrequently termed “androgen independent prostate cancer” or “hormonerefractory prostate cancer,” retains its lineage dependence on ARsignaling.

In certain embodiments, the present invention provides modulators,agonists, and antagonists of AR. In some such embodiments, the AR is anandrogen-resistant AR or an AR mutant associated withcastration-resistant prostate cancer. In some embodiments, the AR has anamino acid sequence as set forth in Table A.

TABLE A Human AR MEVQLGLGRVYPRPPSKTYRGAFQNLFQSVREVIQNPGPRHPEAProtein Sequence ASAAPPGASLLLLQQQQQQQQQQQQQQQQQQQQQETSPRQQQ (Swiss-Prot:QQQGEDGSPQAHRRGPTGYLVLDEEQQPSQPQSALECHPERGC P10275.2)VPEPGAAVAASKGLPQQLPAPPDEDDSAAPSTLSLLGPTFPGLSSCSADLKDILSEASTMQLLQQQQQEAVSEGSSSGRAREASGAPTSSKDNYLGGTSTISDNAKELCKAVSVSMGLGVEALEHLSPGEQLRGDCMYAPLLGVPPAVRPTPCAPLAECKGSLLDDSAGKSTEDTAEYSPFKGGYTKGLEGESLGCSGSAAAGSSGTLELPSTLSLYKSGALDEAAAYQSRDYYNFPLALAGPPPPPPPPHPHARIKLENPLDYGSAWAAAAAQCRYGDLASLHGAGAAGPGSGSPSAAASSSWHTLFTAEEGQLYGPCGGGGGGGGGGGGGGGGGGGGGGGGEAGAVAPYGYTRPPQGLAGQESDFTAPDVWYPGGMVSRVPYPSPTCVKSEMGPWMDSYSGPYGDMRLETARDHVLPIDYYFPPQKTCLICGDEASGCHYGALTCGSCKVFFKRAAEGKQKYLCASRNDCTIDKFRRKNCPSCRLRKCYEAGMTLGARKLKKLGNLKLQEEGEASSTTSPTEETTQKLTVSHIEGYECQPIFLNVLEAIEPGVVCAGHDNNQPDSFAALLSSLNELGERQLVHVVKWAKALPGFRNLHVDDQMAVIQYSWMGLMVFAMGWRSFTNVNSRMLYFAPDLVFNEYRMHKSRMYSQCVRMRHLSQEFGWLQITPQEFLCMKALLLFSIIPVDGLKNQKFFDELRMNYIKELDRIIACKRKNPTSCSRRFYQLTKLLDSVQPIARELHQFTFDLLIKSHMVSVDFPEMMAEIISVQVPKILSGKVKPI YFHTQ (SEQ ID NO: 1)

In some embodiments, an AR modulator as described herein is onedimensioned to fit within the pocket defined by residues F876 and L741of an AR receptor. In some embodiments, an AR modulator as describedherein is one dimensioned to fit within the helix 12 pocket defined as aregion of the AR LBD distal to F876 of an wild type AR receptor. In someembodiments, an AR modulator as described herein is one dimensioned tofit within the helix 12 pocket defined as a region of the AR LBD distalto residue 876 of an F876Xaa mutant AR receptor, wherein Xaa is selectedfrom leucine, isoleucine, tyrosine, cysteine, or serine. In someembodiments, an AR modulator as described herein is one dimensioned tofit within the helix 12 pocket defined as a region of the AR LBD distalto residue L876 of an F876L mutant AR receptor.

In some embodiments, the present invention provides compounds of formulaI:

or a pharmaceutically acceptable salt thereof, wherein:

-   X is CH or N;-   Z is —CH₂— or Ring B; and-   Ring B is an optionally substituted 5-14 membered saturated or    partially unsaturated carbocyclic monocyclic or bicyclic ring,    wherein said ring is spiro-fused at point Z.

As defined generally above, X is CH or N. In some embodiments X is CH.In some embodiments X is N.

As defined generally above, Z is —CH₂— or Ring B.

In some embodiments, Z is —CH₂—. In some embodiments, Z is Ring B.

As defined generally above, Ring B is an optionally substitutedsaturated or partially unsaturated 5-14 membered carbocyclic monocyclicor bicyclic ring, wherein said ring is spiro-fused at point Z. In someembodiments, Ring B is an unsubstituted 5-14 membered saturated orpartially unsaturated carbocyclic monocyclic or bicyclic ring, whereinsaid ring is spiro-fused at point Z. In some embodiments, Ring B is anoptionally substituted 5-8 membered saturated or partially unsaturatedcarbocyclic monocyclic ring, wherein said ring is spiro-fused at pointZ. In some embodiments, Ring B is an optionally substituted 7-14membered saturated or partially unsaturated carbocyclic bicyclic ring,wherein said ring is spiro-fused at point Z. In some embodiments, Ring Bis an optionally substituted saturated 5-8 membered monocyclic ring,wherein said ring is spiro-fused at point Z. In some embodiments, Ring Bis an optionally substituted partially unsaturated 5-8 memberedmonocyclic ring, wherein said ring is spiro-fused at point Z.

One of skill in the art will appreciate that when Ring B isasymmetrically substituted on a tetrahedral carbon, a stereocenterexists. In some embodiments said stereocenter has R stereochemistry. Insome embodiments said stereocenter has S stereochemistry. In someembodiments Ring B is substituted with multiple substituents and eachstereocenter independently has R or S stereochemistry.

In some embodiments, the present invention provides a compound offormula I:

or a pharmaceutically acceptable salt thereof, wherein:

-   X is CH or N;-   Z is —CH₂— or Ring B; and-   Ring B is an 5-14 membered saturated or partially unsaturated    carbocyclic monocyclic or bicyclic ring substituted with n instances    of R^(b), wherein said ring is spiro-fused at point Z;-   n is 0 to 4;    and each R^(b) is independently substituted or unsubstituted C₁₋₆    aliphatic.

In some embodiments, the present invention provides a compound offormula I wherein Z is hydrogen, thereby forming a compound of formulaII:

or a pharmaceutically acceptable salt thereof, wherein X is CH or N.

In some embodiments, the present invention provides a compound offormula I wherein Z is Ring B, thereby forming a compound of formulaIII:

or a pharmaceutically acceptable salt thereof, wherein each of X andRing B is defined above and described in embodiments herein, both singlyand in combination.

In some embodiments, the present invention provides a compound offormula I wherein Z is Ring B, thereby forming a compound of formulaIII:

or a pharmaceutically acceptable salt thereof, wherein each of X, RingB, n and R^(b) is as defined above and described in embodiments herein,both singly and in combination.

In some embodiments, the present invention provides a compound offormula I wherein Z is Ring B, and wherein Ring B is cyclohexylsubstituted with n instances of R^(b), wherein n is 0 to 4 and eachR^(b) is independently substituted or unsubstituted C₁₋₆ aliphatic,thereby forming a compound of formula IV:

wherein X is defined above and described in embodiments herein, bothsingly and in combination.

As defined generally above, each R^(b) is independently substituted orunsubstituted C₁₋₆ aliphatic. In some embodiments, each R^(b) isindependently unsubstituted C₁₋₆ aliphatic. In some embodiments, eachR^(b) is methyl.

As defined generally above, n is 0 to 4. In some embodiments, n is 0. Insome embodiments, n is 1 to 4. In some embodiments, n is 2. In someembodiments, n is 4.

In some embodiments, the present invention provides a compound offormula IV wherein the spiro stereocenter formed between the cyclohexylring and the thiohydantoin ring is in the R configuration or the Sconfiguration, thereby forming a compound of formulae IV-R or IV-S:

wherein each of variables X, n, and R^(b) is defined above and describedin embodiments herein, both singly and in combination.

Exemplary compounds of formula I are set forth in Table 1, below:

TABLE 1 Exemplary Compounds of Formula I Compound ID Compound StructureI-1

I-2

I-3

I-4

I-5

I-6

I-7

I-8

I-9

I-10

I-11

I-12

I-13

I-14

I-15

I-16

I-17

I-18

I-19

I-20

I-21

I-22

I-23

I-24

I-25

I-26

I-27

In certain embodiments, the present invention provides any compoundselected from those depicted in Table 1, above, or a pharmaceuticallyacceptable salt thereof, or any combination of the foregoing.

Compounds or salts thereof provided by the present invention may beutilized in any of a variety of physical forms. For example, in someembodiments, provided compounds (or salts thereof) are utilized in asolid form; in some such embodiments, provided compounds (or saltsthereof) are utilized in an amorphous solid form. In some embodiments,provided compounds are utilized in a crystalline solid form. In someembodiments, provided compounds (or salts thereof) are utilized in asolid form (e.g., a crystalline solid form) that is a solvate orhydrate.

In some embodiments, a composition comprising a compound provided hereincontains only a single physical form of the compound; in someembodiments, a composition comprising a compound provided hereincontains more than one physical form of the compound. In someembodiments, a composition comprising a compound provided hereincontains only a single isomeric (e.g., steroisomer, geometric isomer, orisotopic isomer) form of the compound. In some embodiments, acomposition comprising a compound provided herein contains more than oneisomeric form of the compound.

4. Uses, Formulation and Administration and Pharmaceutically AcceptableCompositions

According to some embodiments, the invention provides a compositioncomprising a compound of this invention or a pharmaceutically acceptablederivative thereof and a pharmaceutically acceptable carrier, adjuvant,or vehicle.

In certain embodiments, the invention provides compositions containingan amount of compound effective to measurably modulate, agonize, orantagonize an AR, in a biological sample or in a patient. In certainembodiments, the amount of compound in compositions of this invention issuch that is effective to measurably modulate, agonize, or antagonize anAR mediated biological process in a biological sample or in a patient.In certain embodiments, provided compositions contain a unit dose amountof a compound described herein, wherein administration of such unit doseamount as part of a therapeutic regimen correlates with a desiredpharmacologic and/or therapeutic outcome.

In certain embodiments, a composition of this invention is formulatedfor administration to a patient in need of such composition. In someembodiments, a composition of this invention is formulated for oraladministration to a patient.

As used herein, a “dosing regimen” or “therapeutic regimen” refers to aset of unit doses (typically more than one) that are administeredindividually to a subject, typically separated by periods of time. Insome embodiments, a given therapeutic agent has a recommended dosingregimen, which may involve one or more doses. In some embodiments, adosing regimen comprises a plurality of doses each of which areseparated from one another by a time period of the same length; in someembodiments, a dosing regime comprises a plurality of doses and at leasttwo different time periods separating individual doses. In someembodiments, all doses within a dosing regimen are of the same unit doseamount. In some embodiments, different doses within a dosing regimen areof different amounts. In some embodiments, a dosing regimen comprises afirst dose in a first dose amount, followed by one or more additionaldoses in a second dose amount different from the first dose amount. Insome embodiments, a dosing regimen comprises a first dose in a firstdose amount, followed by one or more additional doses in a second doseamount same as the first dose amount.

The term “patient,” as used herein, means an animal, often a mammal, andin many embodiments a human.

The term “pharmaceutically acceptable carrier, adjuvant, or vehicle”, asused herein, refers to a non-toxic carrier, adjuvant, or vehicle thatdoes not destroy the pharmacological activity of the compound with whichit is formulated. Pharmaceutically acceptable carriers, adjuvants orvehicles that may be used in the compositions of this invention include,but are not limited to, ion exchangers, alumina, aluminum stearate,lecithin, serum proteins, such as human serum albumin, buffer substancessuch as phosphates, glycine, sorbic acid, potassium sorbate, partialglyceride mixtures of saturated vegetable fatty acids, water, salts orelectrolytes, such as protamine sulfate, disodium hydrogen phosphate,potassium hydrogen phosphate, sodium chloride, zinc salts, colloidalsilica, magnesium trisilicate, polyvinyl pyrrolidone, cellulose-basedsubstances, polyethylene glycol, sodium carboxymethylcellulose,polyacrylates, waxes, polyethylene-polyoxypropylene-block polymers,polyethylene glycol and wool fat.

A “pharmaceutically acceptable derivative” means any non-toxic salt,ester, or salt of an ester of a compound of this invention that, uponadministration to a recipient, is capable of providing, either directlyor indirectly, a compound of this invention or an inhibitorily activemetabolite or residue thereof.

As used herein, the term “active metabolite or residue thereof” meansthat a metabolite or residue thereof is also an agonist or antagonist ofAR or is retains therapeutic activity in treating the same disease,disorder or condition.

Compositions of the present invention may be formulated for anyappropriate route of administration. For example, in some embodiments,provided compositions may be administered orally, parenterally, byinhalation spray, topically, rectally, nasally, buccally, vaginally orvia an implanted reservoir. The term “parenteral” as used hereinincludes subcutaneous, intravenous, intramuscular, intra-articular,intra-synovial, intrasternal, intrathecal, intrahepatic, intralesionaland intracranial injection or infusion techniques. In some embodiments,provided compositions are administered orally, intraperitoneally orintravenously. Sterile injectable forms of the compositions of thisinvention may be aqueous or oleaginous suspension. Such suspensions maybe formulated according to techniques known in the art using suitabledispersing or wetting agents and suspending agents.

In some embodiments, pharmaceutically acceptable compositions of theinvention may be formulated as injectable preparations. Injectablepreparations, for example, sterile injectable aqueous or oleaginoussuspensions may be formulated according to the known art using suitabledispersing or wetting agents and suspending agents. The sterileinjectable preparation may also be a sterile injectable solution,suspension or emulsion in a nontoxic parenterally acceptable diluent orsolvent, for example, as a solution in 1,3-butanediol. Among theacceptable vehicles and solvents that may be employed are water,Ringer's solution, U.S.P. and isotonic sodium chloride solution. Inaddition, sterile, fixed oils are conventionally employed as a solventor suspending medium. For this purpose any bland fixed oil can beemployed including synthetic mono- or diglycerides. In addition, fattyacids such as oleic acid are used in the preparation of injectables.

In some embodiments, injectable formulations can be sterilized, forexample, by filtration through a bacterial-retaining filter, or byincorporating sterilizing agents in the form of sterile solidcompositions which can be dissolved or dispersed in sterile water orother sterile injectable medium prior to use.

In some embodiments, for example in order to prolong effects of acompound or composition, it may be desirable to slow the absorption ofthe compound from subcutaneous or intramuscular injection. This may beaccomplished by the use of a liquid suspension of crystalline oramorphous material with poor water solubility. The rate of absorption ofthe compound then depends upon its rate of dissolution that, in turn,may depend upon crystal size and crystalline form. Alternatively oradditionally, delayed absorption of a parenterally administered compoundform is accomplished by dissolving or suspending the compound in an oilvehicle. Injectable depot forms are made by forming microencapsulematrices of the compound in biodegradable polymers such aspolylactide-polyglycolide. Depending upon the ratio of compound topolymer and the nature of the particular polymer employed, the rate ofcompound release can be controlled. Examples of other biodegradablepolymers include poly(orthoesters) and poly(anhydrides). Depotinjectable formulations are also prepared by entrapping the compound inliposomes or microemulsions that are compatible with body tissues.

In some embodiments, sterile injectable preparations may be or include asterile injectable solution or suspension in a non-toxic parenterallyacceptable diluent or solvent, for example as a solution in1,3-butanediol. Among the acceptable vehicles and solvents that may beemployed are water, Ringer's solution and isotonic sodium chloridesolution. In addition, sterile, fixed oils are conventionally employedas a solvent or suspending medium.

For this purpose, any bland fixed oil may be employed includingsynthetic mono- or di-glycerides. Fatty acids, such as oleic acid andits glyceride derivatives are useful in the preparation of injectables,as are natural pharmaceutically-acceptable oils, such as olive oil orcastor oil, especially in their polyoxyethylated versions. Such oilsolutions or suspensions may also contain a long-chain alcohol diluentor dispersant, such as carboxymethyl cellulose or similar dispersingagents that are commonly used in the formulation of pharmaceuticallyacceptable dosage forms including emulsions and suspensions. Othercommonly used surfactants, such as Tweens, Spans and other emulsifyingagents or bioavailability enhancers which are commonly used in themanufacture of pharmaceutically acceptable solid, liquid, or otherdosage forms may also be used for the purposes of formulation.

Pharmaceutically acceptable compositions of this invention may be orallyadministered in any orally acceptable dosage form including, but notlimited to, capsules, tablets, aqueous suspensions or solutions. In thecase of tablets for oral use, carriers commonly used include lactose andcorn starch. Lubricating agents, such as magnesium stearate, are alsotypically added. For oral administration in a capsule form, usefuldiluents include lactose and dried cornstarch. When aqueous suspensionsare required for oral use, the active ingredient is combined withemulsifying and suspending agents. If desired, certain sweetening,flavoring or coloring agents may also be added.

Solid dosage forms for oral administration include capsules, tablets,pills, powders, and granules. In such solid dosage forms, the activecompound is mixed with at least one inert, pharmaceutically acceptableexcipient or carrier such as sodium citrate or dicalcium phosphateand/or a) fillers or extenders such as starches, lactose, sucrose,glucose, mannitol, and silicic acid, b) binders such as, for example,carboxymethylcellulose, alginates, gelatin, polyvinylpyrrolidinone,sucrose, and acacia, c) humectants such as glycerol, d) disintegratingagents such as agar-agar, calcium carbonate, potato or tapioca starch,alginic acid, certain silicates, and sodium carbonate, e) solutionretarding agents such as paraffin, f) absorption accelerators such asquaternary ammonium compounds, g) wetting agents such as, for example,cetyl alcohol and glycerol monostearate, h) absorbents such as kaolinand bentonite clay, and i) lubricants such as talc, calcium stearate,magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate,and mixtures thereof. In the case of capsules, tablets and pills, thedosage form may also comprise buffering agents.

Solid compositions of a similar type may also be employed as fillers insoft and hard-filled gelatin capsules using such excipients as lactoseor milk sugar as well as high molecular weight polyethylene glycols andthe like. The solid dosage forms of tablets, dragees, capsules, pills,and granules can be prepared with coatings and shells such as entericcoatings and other coatings well known in the pharmaceutical formulatingart. They may optionally contain opacifying agents and can also be of acomposition that they release the active ingredient(s) only, orpreferentially, in a certain part of the intestinal tract, optionally,in a delayed manner. Examples of embedding compositions that can be usedinclude polymeric substances and waxes. Solid compositions of a similartype may also be employed as fillers in soft and hard-filled gelatincapsules using such excipients as lactose or milk sugar as well as highmolecular weight polethylene glycols and the like.

In some embodiments, provided compounds can be in micro-encapsulatedform with one or more excipients as noted above. Solid dosage forms suchas tablets, dragees, capsules, pills, and granules can be prepared withcoatings and shells such as enteric coatings, release controllingcoatings and other coatings well known in the pharmaceutical formulatingart. In such solid dosage forms, the active compound may be admixed withat least one inert diluent such as sucrose, lactose or starch. Suchdosage forms may also comprise, as is normal practice, additionalsubstances other than inert diluents, e.g., tableting lubricants andother tableting aids such a magnesium stearate and microcrystallinecellulose. In the case of capsules, tablets and pills, the dosage formsmay also comprise buffering agents. They may optionally containopacifying agents and can also be of a composition that they release theactive ingredient(s) only, or preferentially, in a certain part of theintestinal tract, optionally, in a delayed manner. Examples of embeddingcompositions that can be used include polymeric substances and waxes.

Liquid dosage forms for oral administration include, but are not limitedto, pharmaceutically acceptable emulsions, microemulsions, solutions,suspensions, syrups and elixirs. In addition to the active compounds,the liquid dosage forms may contain inert diluents commonly used in theart such as, for example, water or other solvents, solubilizing agentsand emulsifiers such as ethyl alcohol, isopropyl alcohol, ethylcarbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propyleneglycol, 1,3-butylene glycol, dimethylformamide, oils (in particular,cottonseed, groundnut, corn, germ, olive, castor, and sesame oils),glycerol, tetrahydrofurfuryl alcohol, polyethylene glycols and fattyacid esters of sorbitan, and mixtures thereof. Besides inert diluents,the oral compositions can also include adjuvants such as wetting agents,emulsifying and suspending agents, sweetening, flavoring, and perfumingagents.

Alternatively or additionally, pharmaceutically acceptable compositionsof this invention may be administered in the form of suppositories forrectal administration. Such compositions can be prepared by combining aprovided compound with a suitable non-irritating excipient that is solidat room temperature but liquid at rectal temperature and therefore willmelt in the rectum to release the drug. Such materials include cocoabutter, beeswax and polyethylene glycols.

In some embodiments, pharmaceutically acceptable compositions of thisinvention may be administered topically, especially when the target oftreatment includes areas or organs readily accessible by topicalapplication, including diseases of the eye, the skin, or the lowerintestinal tract. Suitable topical formulations are readily prepared foreach of these areas or organs.

Topical application for the lower intestinal tract can be effected in arectal suppository formulation (see above) or in a suitable enemaformulation. Topically-transdermal patches may also be used.

For topical applications, provided pharmaceutically acceptablecompositions may be formulated in a suitable ointment containing theactive component suspended or dissolved in one or more carriers.Carriers for topical administration of compounds of this inventioninclude, but are not limited to, mineral oil, liquid petrolatum, whitepetrolatum, propylene glycol, polyoxyethylene, polyoxypropylenecompound, emulsifying wax and water. Alternatively or additionally,provided pharmaceutically acceptable compositions can be formulated in asuitable lotion or cream containing the active components suspended ordissolved in one or more pharmaceutically acceptable carriers. Suitablecarriers include, but are not limited to, mineral oil, sorbitanmonostearate, polysorbate 60, cetyl esters wax, cetearyl alcohol,2-octyldodecanol, benzyl alcohol and water.

Dosage forms for topical or transdermal administration of a compound ofthis invention include ointments, pastes, creams, lotions, gels,powders, solutions, sprays, inhalants or patches. The active componentis admixed under sterile conditions with a pharmaceutically acceptablecarrier and any needed preservatives or buffers as may be required.Ophthalmic formulation, ear drops, and eye drops are also contemplatedas being within the scope of this invention. Additionally, the presentinvention contemplates the use of transdermal patches, which have theadded advantage of providing controlled delivery of a compound to thebody. Such dosage forms can be made by dissolving or dispensing thecompound in the proper medium. Absorption enhancers can also be used toincrease the flux of the compound across the skin. The rate can becontrolled by either providing a rate controlling membrane or bydispersing the compound in a polymer matrix or gel.

For ophthalmic use, provided pharmaceutically acceptable compositionsmay be formulated as micronized suspensions in isotonic, pH adjustedsterile saline, or, preferably, as solutions in isotonic, pH adjustedsterile saline, either with or without a preservative such asbenzylalkonium chloride. Alternatively or additionally, for ophthalmicuses, the pharmaceutically acceptable compositions may be formulated inan ointment such as petrolatum.

In some embodiments, pharmaceutically acceptable compositions of thisinvention may be administered by nasal aerosol or inhalation. Suchcompositions may be prepared according to techniques well-known in theart of pharmaceutical formulation, for example as solutions in saline,employing benzyl alcohol or other suitable preservatives, absorptionpromoters to enhance bioavailability, fluorocarbons, and/or otherconventional solubilizing or dispersing agents.

In some embodiments, pharmaceutically acceptable compositions of thisinvention are formulated for oral administration. Such formulations maybe administered with or without food. In some embodiments,pharmaceutically acceptable compositions of this invention areadministered without food. In some embodiments, pharmaceuticallyacceptable compositions of this invention are administered with food.

The amount of compounds of the present invention that may be combinedwith the carrier materials to produce a composition in a single dosageform will vary depending upon the host treated, the particular mode ofadministration. In some embodiments provided compositions are formulatedso that a dosage of between 0.01-100 mg/kg body weight/day of theinhibitor can be administered to a patient receiving these compositions.

It should also be understood that a specific dosage and treatmentregimen for any particular patient may depend upon a variety of factors,including the activity of the specific compound employed, the age, bodyweight, general health, sex, diet, time of administration, rate ofexcretion, drug combination, and the judgment of the treating physicianand the severity of the particular disease being treated. In someembodiments, amount of a compound of the present invention included in acomposition described herein is determined by activity and/orbioavailability of the particular compound, so that compositions ofdifferent compounds may include different absolute amounts of compound.

Uses of Compounds and Pharmaceutically Acceptable Compositions

Compounds and compositions described herein are useful in the treatmentof any of a variety of diseases, disorders, and conditions. In someembodiments, provided compounds and compositions are useful in thetreatment of diseases, disorders, or conditions associated with activityof androgen receptors.

The activity of a compound utilized in this invention as a modulator,agonist or antagonist of AR or treatment for an AR-mediated disease,disorder or condition, may be assayed in vitro or in vivo. An in vivoassessment of the efficacy of the compounds of the invention may be madeusing an animal model of an AR-mediated disease, disorder or condition,e.g., a rodent or primate model. Cell-based assays may be performedusing, e.g., a cell line isolated from a tissue that expresses eitherwild type or mutant AR. Additionally, biochemical or mechanism-basedassays, e.g., transcription assays using a purified protein, Northernblot, RT-PCR, etc., may be performed. In vitro assays include assaysthat determine cell morphology, protein expression, and/or thecytotoxicity, enzyme inhibitory activity, and/or the subsequentfunctional consequences of treatment of cells with compounds of theinvention. Alternate or additional in vitro assays may be used toquantitate the ability of the inhibitor to bind to protein or nucleicacid molecules within the cell. Inhibitor binding may be measured byradiolabelling the inhibitor prior to binding, isolating theinhibitor/target molecule complex and determining the amount ofradiolabel bound. Alternatively or additionally, inhibitor binding maybe determined by running a competition experiment where new inhibitorsare incubated with purified proteins or nucleic acids bound to knownradioligands. Detailed conditions of exemplary systems for assaying acompound utilized in this invention as a modulator, agonist orantagonist of AR are set forth in the Examples below. Such assays areexemplary and not intended to limit the scope of the invention. Theskilled practitioner can appreciate that modifications can be made toconventional assays to develop equivalent or other assays that can beemployed to comparably assess activity or otherwise characterizecompounds and/or compositions as described herein.

As used herein, the terms “treatment,” “treat,” and “treating” refer toreversing, alleviating, delaying the onset of, reducing incidence orseverity, or inhibiting the progress of a disease, disorder orcondition, or one or more symptoms thereof, as described herein. In someembodiments, treatment may be administered after one or more symptomshave developed. In other embodiments, treatment may be administered inthe absence of symptoms. For example, treatment may be administered to asusceptible individual prior to the onset of symptoms (e.g., in light ofa history of symptoms and/or in light of genetic or other susceptibilityfactors). Treatment may also be continued after symptoms have resolved,for example to prevent or delay their recurrence.

Compounds and/or compositions described herein may be administered usingany amount and any route of administration effective for treating adisease, disorder, or condition. In some embodiments, compounds and/orcompostions are administered in an amount and/or by a route effectivefor treating a cardiovascular disease, disorder or condition, aninflammatory disease, disorder or condition, a neurological disease,disorder or condition, an ocular disease, disorder or condition, ametabolic disease, disorder or condition, a cancer or otherproliferative disease, disorder or condition, a reproductive disease,disorder or condition, or a bone disease, disorder or condition.

In some embodiments, compounds and/or compositions described herein maybe administered using any amount and any route of administrationeffective for treating or lessening the severity of a disease, disorderor condition associated with AR.

In some embodiments, the compounds and compositions, according to themethod of the present invention, may be administered using any amountand any route of administration effective for treating a cancer oranother proliferative disease, disorder or condition. In someembodiments, the cancer or other proliferative disease, disorder orcondition is a prostate cancer. In some embodiments, the cancer or otherproliferative disease, disorder or condition is a castration-resistantprostate cancer (CRPC). In some embodiments, the cancer or otherproliferative disease, disorder or condition is a castration-resistantprostate cancer (CRPC) bearing a mutation in AR. In some embodiments,the mutation in AR is a mutation of Phe876. In some embodiments, themutation in AR is a mutation of Phe876 to leucine. In some embodiments,the mutation in AR is a mutation of Phe876 to isoleucine. In someembodiments, the mutation in AR is a mutation of Phe876 to valine. Insome embodiments, the mutation in AR is a mutation of Phe876 to serine.In some embodiments, the mutation in AR is a mutation of Phe876 tocysteine. In some embodiments, the mutation in AR is a mutation ofPhe876 to tyrosine. In some embodiments, the cancer or otherproliferative disease, disorder or condition is a prostate cancer thatis resistant to treatment with Enzalutamide.

The present invention encompasses the recognition that mutations in theAR polypeptide can render the AR polypeptide resistant to anti-androgensor convert anti-androgens to androgen agonists. In some embodiments, theinvention provides compounds that can be used to effect anti-androgeniceffects despite the presence of such mutations.

The amino acid sequence of an AR polypeptide described herein can existin a mutant AR containing, or can be modified to produce an mutant ARpolypeptide variant at least one (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,or more) additions, substitutions, or deletions of a wild-type aminoacid residue.

In some embodiments, the AR polypeptide variants described herein resultin a loss of inhibition of AR activity by one or more antiandrogens of0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 up to 100%. In some embodiments, the ARpolypeptide variants described herein convert anti-androgens to androgenreceptor agonists.

Specific, nonlimiting amino acid residues that can be modified in an ARmutant include, e.g., E566, E589, E669, C687, A700, N772, H777, C785,F877, K911, of the AR polypeptide. These amino acid residues can besubstituted with any amino acid or amino acid analog. For example, thesubstitutions at the recited positions can be made with any of thenaturally-occurring amino acids (e.g., alanine, aspartic acid,asparagine, arginine, cysteine, glycine, glutamic acid, glutamine,histidine, leucine, valine, isoleucine, lysine, methionine, proline,threonine, serine, phenylalanine, tryptophan, or tyrosine). Inparticular instances, an amino acid substitution is E566K, E589K, E669K,C687Y, A700T, N772S, H777Y, C785R, F877C, F877I, F877L, F877S, F877V,F877Y and/or K911E.

In some embodiments, the AR mutants as described herein can includeadditional modifications of the AR polypeptide previously described inthe art, including but not limited to, e.g., A597T, S648G, P683T, D696E,R727H, N728I, I738F, W741L, W741C, W741L, M743V, G751S, A871V, H874Y,T878A, T878S, and P914S.

In some embodiments, the compounds and compositions, according to themethod of the present invention, may be administered using any amountand any route of administration effective for treating a bone disease,disorder or condition. In some embodiments, the bone disease, disorderor condition is osteoporosis.

In will be appreciated by those skilled in the art that the exact amountof a provided compound or composition may vary from subject to subject,depending on the species, age, and general condition of the subject, theseverity of the infection, the particular agent, its mode ofadministration, and the like.

In some embodiments, compounds of the invention are formulated in dosageunit form, for example for ease of administration and uniformity ofdosage. The expression “dosage unit form” or “unit dosage” as usedherein refers to a physically discrete unit of agent appropriate for thepatient to be treated. It will be understood, however, that total dailyusage of the compounds and compositions of the present invention may bedecided by the attending physician within the scope of sound medicaljudgment. The specific effective dose level for any particular patientor organism may depend upon a variety of factors including the disorderbeing treated and the severity of the disorder; the activity of thespecific compound employed; the specific composition employed; the age,body weight, general health, sex and diet of the patient; the time ofadministration, route of administration, and rate of excretion of thespecific compound employed; the duration of the treatment; drugs used incombination or coincidental with the specific compound employed, andlike factors well known in the medical arts.

According to some embodiments, the invention relates to a method ofmodulating, agonizing or antagonizing AR in a biological samplecomprising the step of contacting said biological sample with a compoundof this invention, or a composition comprising said compound. In someembodiments, the invention provides a method of antagonizing AR in abiological sample comprising the step of contacting said biologicalsample with a compound of this invention. In some embodiments, theinvention provides a method of agonizing AR in a biological samplecomprising the step of contacting said biological sample with a compoundof this invention. In some embodiments, the agonism of the AR is partialagonism.

The term “biological sample”, as used herein, includes, withoutlimitation, cell cultures or extracts thereof; biopsied materialobtained from a mammal or extracts thereof; and blood, saliva, urine,feces, semen, tears, or other body fluids or extracts thereof.

Agonism or antagonism of receptors in a biological sample is useful fora variety of purposes that are known to one of skill in the art.Examples of such purposes include, but are not limited to biologicalassays, gene expression studies, and biological target identification.

Some embodiments of the present invention relate to a method ofmodulating, agonizing or antagonizing AR in a patient comprising thestep of administering to said patient a compound of the presentinvention, or a composition comprising said compound. In someembodiments the invention provides a method of antagonizing AR in apatient comprising the step of administering to said patient a compoundof the present invention, or a composition comprising said compound.

In some embodiments, the invention relates to a method of modulating,agonizing or antagonizing AR activity in a patient comprising the stepof administering to said patient a compound of the present invention, ora composition comprising said compound. In certain embodiments, thepresent invention provides a method for treating a disease, disorder orcondition mediated by AR, in a patient in need thereof, comprising thestep of administering to said patient a compound according to thepresent invention or pharmaceutically acceptable composition thereof.Such diseases, disorders and conditions are described in detail herein.

In some embodiments compounds and/or compositions of the presentinvention may be used in a method of treating a cardiovascular disease,disorder, or condition, an inflammatory disease, disorder or condition,a neurological disease, disorder or condition, an ocular disease,disorder or condition, a metabolic disease, disorder or condition, acancer or other proliferative disease, disorder or condition, areproductive disease, disorder or condition, or a bone disease, disorderor condition. In certain embodiments the compounds and compositions ofthe present invention may be used to treat a cardiovascular disease,disorder or condition, an inflammatory disease, disorder or condition, aneurological disease, disorder or condition, an ocular disease, disorderor condition, a metabolic disease, disorder or condition, a cancer orother proliferative disease, disorder or condition, a reproductivedisease, disorder or condition, or a bone disease, disorder or conditionin a mammal. In certain embodiments the mammal is a human patient.

In some embodiments the present invention provides a method of treatinga cardiovascular disease, disorder or condition, an inflammatorydisease, disorder or condition, a neurological disease, disorder orcondition, an ocular disease, disorder or condition, a metabolicdisease, disorder or condition, a cancer or other proliferative disease,disorder or condition, a reproductive disease, disorder or condition, ora bone disease, disorder or condition, comprising administering acompound or composition of the present invention to a patient in needthereof. In certain embodiments the method of treating a cardiovasculardisease, disorder or condition, an inflammatory disease, disorder orcondition, a neurological disease, disorder or condition, an oculardisease, disorder or condition, a metabolic disease, disorder orcondition, a cancer or other proliferative disease, disorder orcondition, a reproductive disease, disorder or condition, or a bonedisease, disorder or condition comprises administering compounds andcompositions of the present invention to a mammal. In certainembodiments the mammal is a human.

In certain embodiments, the present invention provides a method oftreating a cancer or another proliferative disease, disorder orcondition, comprising administering a compound or composition of thepresent invention to a patient with a cancer or another proliferativedisease, disorder or condition. In certain embodiments, the method oftreating a cancer or other proliferative disorder comprisesadministering compounds and compositions of the present invention to amammal. In certain embodiments, the cancer or other proliferativedisorder is a prostate cancer. In certain embodiments, the prostatecancer is a castration-resistant prostate cancer. In certainembodiments, the mammal is a human.

As used herein, the terms “treating a cancer” refers to the inhibitionof the growth, division, maturation or viability of cancer cells, and/orcausing the death of cancer cells, individually or in aggregate withother cancer cells, by cytotoxicity, nutrient depletion, or theinduction of apoptosis.

Examples of tissues containing cancerous cells whose proliferation isinhibited by the compounds and compositions described herein and againstwhich the methods described herein are useful include but are notlimited to breast, prostate, brain, blood, bone marrow, bone, liver,pancreas, skin, kidney, colon, ovary, lung, testicle, penis, thyroid,parathyroid, pituitary, thymus, retina, uvea, conjunctiva, spleen, head,neck, trachea, gall bladder, rectum, salivary gland, adrenal gland,throat, esophagus, lymph nodes, sweat glands, sebaceous glands, muscle,heart, and stomach.

In some embodiments, the cancer treated by compounds or compositions ofthe invention is a skin cancer, lung cancer, breast cancer, prostatecancer, leukemia, kidney cancer, esophageal cancer, brain cancer, bonecancer or colon cancer. In some embodiments, the cancer treated by thecompounds or compositions of the invention is a prostate cancer.

Depending upon the particular disease, disorder or condition to betreated, additional therapeutic agents, which are normally administeredto treat that condition, may be administered in combination withcompounds and compositions of this invention. As used herein, additionaltherapeutic agents that are normally administered to treat a particulardisease, or condition, are known as “appropriate for the disease, orcondition, being treated”.

In certain embodiments, a provided compound, or composition thereof, isadministered in combination with another modulator, agonist orantagonist of AR. In some embodiments, a provided compound, orcomposition thereof, is administered in combination with one or moreother therapeutic agents. In some embodiments the AR modulators,agonists or antagonists include, but are not limited to non-steroidalantiandrogens, aminoglutethimide, enzalutamide, bicalutamide,nilutamide, flutamide, steroidal antiandrogens, finasteride,dutasteride, bexlosteride, izonsteride, turosteride, epristeride, otherinhibitors of 5-alpha-reductase, 3,3′-diindolylmethane (DIM),N-butylbenzene-sulfonamide (NBBS).

In certain embodiments, a provided compound, or composition thereof, isadministered in combination with another therapeutic agent, wherein saidtherapeutic agent is a an androgen deprivation therapeutic agent. Insome embodiments, the androgen deprivation therapeutic agent isabiraterone, abiraterone acetate, buserelin, cyproterone, cyproteroneacetate, degarelix, goserelin, ketoconazole, Lupron (leoprorelin,leuprolide acetate), orteronel (TAK-700), spironolactone, ortriptorelin.

In certain embodiments, a provided compound, or a composition thereof,is administered in combination with another anti-cancer, cytotoxin,chemotherapeutic agent, or radiotherapeutic agent, and/or with anotheragent (e.g., a palliative agent, pain reliever, anti-emetic agent,anti-nausea agent, anti-inflammatory agent, etc.) commonly administeredto cancer patients.

In certain embodiments, the anti-cancer or chemotherapeutic agents usedin combination with compounds or compositions of the invention include,but are not limited to imatinib, nilotinib, gefitinib, sunitinib,carfilzomib, salinosporamide A, retinoic acid, cisplatin, carboplatin,oxaliplatin, mechlorethamine, cyclophosphamide, chlorambucil,ifosfamide, azathioprine, mercaptopurine, doxifluridine, fluorouracil,gemcitabine, methotrexate, tioguanine, vincristine, vinblastine,vinorelbine, vindesine, podophyllotoxin, etoposide, teniposide,tafluposide, paclitaxel, docetaxel, irinotecan, topotecan, amsacrine,actinomycin, doxorubicin, daunorubicin, valrubicin, idarubicin,epirubicin, plicamycin, mitomycin, mitoxantrone, melphalan, busulfan,capecitabine, pemetrexed, epothilones, 13-cis-Retinoic Acid, 2-CdA,2-Chlorodeoxyadenosine, 5-Azacitidine, 5-Fluorouracil, 5-FU,6-Mercaptopurine, 6-MP, 6-TG, 6-Thioguanine, Abraxane, Accutane®,Actinomycin-D, Adriamycin®, Adrucil®, Afinitor®, Agrylin®, Ala-Cort®,Aldesleukin, Alemtuzumab, ALIMTA, Alitretinoin, Alkaban-AQ®, Alkeran®,All-transretinoic Acid, Alpha Interferon, Altretamine, Amethopterin,Amifostine, Aminoglutethimide, Anagrelide, Anandron®, Anastrozole,Arabinosylcytosine, Ara-C, Aranesp®, Aredia®, Arimidex®, Aromasin®,Arranon®, Arsenic Trioxide, Arzerra™, Asparaginase, ATRA, Avastin®,Azacitidine, BCG, BCNU, Bendamustine, Bevacizumab, Bexarotene, BEXXAR®,Bicalutamide, BiCNU, Blenoxane®, Bleomycin, Bortezomib, Busulfan,Busulfex®, C225, Calcium Leucovorin, Campath®, Camptosar®,Camptothecin-11, Capecitabine, Carac™, Carboplatin, Carmustine,Carmustine Wafer, Casodex®, CC-5013, CCI-779, CCNU, CDDP, CeeNU,Cerubidine®, Cetuximab, Chlorambucil, Citrovorum Factor, Cladribine,Cortisone, Cosmegen®, CPT-11, Cytadren®, Cytosar-U®, Cytoxan®,Dacarbazine, Dacogen, Dactinomycin, Darbepoetin Alfa, Dasatinib,Daunomycin, Daunorubicin Hydrochloride, Daunorubicin Liposomal,DaunoXome®, Decadron, Decitabine, Delta-Cortef®, Deltasone®, Denileukin,Diftitox, DepoCyt™, Dexamethasone, Dexamethasone Acetate, DexamethasoneSodium Phosphate, Dexasone, Dexrazoxane, DHAD, DIC, Diodex, Docetaxel,Doxil®, Doxorubicin, Doxorubicin Liposomal, Droxia™, DTIC, DTIC-Dome®,Duralone®, Efudex®, Eligard™, Ellence™, Eloxatin™, Elspar®, Emcyt®,Epirubicin, Epoetin Alfa, Erbitux, Erlotinib, Erwinia L-asparaginase,Estramustine, Ethyol, Etopophos®, Etoposide, Etoposide Phosphate,Eulexin®, Everolimus, Evista®, Exemestane, Fareston®, Faslodex®,Femara®, Filgrastim, Floxuridine, Fludara®, Fludarabine, Fluoroplex®,Fluorouracil, Fluorouracil (cream), Fluoxymesterone, Flutamide, FolinicAcid, FUDR®, Fulvestrant, G-CSF, Gefitinib, Gemcitabine, Gemtuzumab,ozogamicin, Gemzar Gleevec™, Gliadel® Wafer, GM-CSF, Goserelin,Granulocyte—Colony Stimulating Factor, Granulocyte Macrophage ColonyStimulating Factor, Halotestin®, Herceptin®, Hexadrol, Hexalen®,Hexamethylmelamine, HMM, Hycamtin®, Hydrea®, Hydrocort Acetate®,Hydrocortisone, Hydrocortisone Sodium Phosphate, Hydrocortisone SodiumSuccinate, Hydrocortone Phosphate, Hydroxyurea, Ibritumomab,Ibritumomab, Tiuxetan, Idamycin®, Idarubicin Ifex®, IFN-alpha,Ifosfamide, IL-11, IL-2, Imatinib mesylate, Imidazole Carboxamide,Interferon alfa, Interferon Alfa-2b (PEG Conjugate), Interleukin-2,Interleukin-11, Intron A® (interferon alfa-2b), Iressa®, Irinotecan,Isotretinoin, Ixabepilone, Ixempra™, Kidrolase®, Lanacort®, Lapatinib,L-asparaginase, LCR, Lenalidomide, Letrozole, Leucovorin, Leukeran,Leukine™, Leuprolide, Leurocristine, Leustatin™, Liposomal Ara-C, LiquidPred®, Lomustine, L-PAM, L-Sarcolysin, Lupron®, Lupron Depot®,Matulane®, Maxidex, Mechlorethamine, Mechlorethamine Hydrochloride,Medralone®, Medrol®, Megace®, Megestrol, Megestrol Acetate, Melphalan,Mercaptopurine, Mesna, Mesnex™, Methotrexate, Methotrexate Sodium,Methylprednisolone, Meticorten®, Mitomycin, Mitomycin-C, Mitoxantrone,M-Prednisol®, MTC, MTX, Mustargen®, Mustine, Mutamycin®, Myleran®,Mylocel™, Mylotarg®, Navelbine®, Nelarabine, Neosar®, Neulasta™,Neumega®, Neupogen®, Nexavar®, Nilandron®, Nilotinib, Nilutamide,Nipent®, Nitrogen Mustard, Novaldex®, Novantrone®, Nplate, Octreotide,Octreotide acetate, Ofatumumab, Oncospar®, Oncovin®, Ontak®, Onxal™,Oprelvekin, Orapred®, Orasone®, Oxaliplatin, Paclitaxel, PaclitaxelProtein-bound, Pamidronate, Panitumumab, Panretin®, Paraplatin®,Pazopanib, Pediapred®, PEG Interferon, Pegaspargase, Pegfilgrastim,PEG-INTRON™, PEG-L-asparaginase, PEMETREXED, Pentostatin, PhenylalanineMustard, Platinol®, Platinol-AQ®, Prednisolone, Prednisone, Prelone®,Procarbazine, PROCRIT®, Proleukin®, Prolifeprospan 20 with CarmustineImplant, Purinethol®, Raloxifene, Revlimid®, Rheumatrex®, Rituxan®,Rituximab, Roferon-A® (Interferon Alfa-2a), Romiplostim, Rubex®,Rubidomycin hydrochloride, Sandostatin®, Sandostatin LAR®, Sargramostim,Solu-Cortef®, Solu-Medrol®, Sorafenib, SPRYCEL™, STI-571, Streptozocin,SU11248, Sunitinib, Sutent®, Tamoxifen, Tarceva®, Targretin®, Tasigna®,Taxol®, Taxotere®, Temodar®, Temozolomide, Temsirolimus, Teniposide,TESPA, Thalidomide, Thalomid®, TheraCys®, Thioguanine, ThioguanineTabloid®, Thiophosphoamide, Thioplex®, Thiotepa, TICE®, Toposar®,Topotecan, Toremifene, Torisel®, Tositumomab, Trastuzumab, Treanda®,Tretinoin, Trexall™, Trisenox®, TSPA, TYKERB®, VCR, Vectibix™, Velban®,Velcade®, VePesid®, Vesanoid®, Viadur™, Vidaza®, Vinblastine,Vinblastine Sulfate, Vincasar Pfs®, Vincristine, Vinorelbine,Vinorelbine tartrate, VLB, VM-26, Vorinostat, Votrient, VP-16, Vumon®,Xeloda®, Zanosar®, Zevalin™, Zinecard®, Zoladex®, Zoledronic acid,Zolinza, Zometa®, or combinations of any of the above.

In some embodiments, a provided compound, or a composition thereof, isadministered to a patient undergoing radiation therapy. In someembodiments, the radiation therapy is external beam gamma or x-rayradiation therapy. In some embodiments, the radiation therapy isbrachytherapy. In some embodiments, the brachytherapy uses ¹⁹⁸Au, ²⁵²Cf,⁶⁰Co, ¹³⁷Cs, ¹²⁵I, ¹⁹²Ir, ³²P, ¹⁰³Pd, ²²⁶Ra, ¹⁰⁶Ru, ¹⁴⁵Sm, ⁹⁰Sr, and¹⁸²Ta.

In certain embodiments, a combination of 2 or more therapeutic agentsmay be administered together with compounds of the invention. In certainembodiments, a combination of 3 or more therapeutic agents may beadministered with compounds of the invention.

Other examples of agents the inhibitors of this invention may also becombined with include, without limitation: vitamins and nutritionalsupplements, cancer vaccines, treatments for neutropenia (e.g. G-CSF,filgrastim, lenograstim), treatments for thrombocytopenia (e.g. bloodtransfusion, erythropoietin), antiemetics (e.g. 5-HT₃ receptorantagonists, dopamine antagonists, NK1 receptor antagonists, histaminereceptor antagonists, cannabinoids, benzodiazepines, oranticholinergics), treatments for Alzheimer's Disease such as Aricept®and Excelon®; treatments for Parkinson's Disease such asL-DOPA/carbidopa, entacapone, ropinrole, pramipexole, bromocriptine,pergolide, trihexephendyl, and amantadine; agents for treating MultipleSclerosis (MS) such as beta interferon (e.g., Avonex® and Rebif®),Copaxone®, and mitoxantrone; treatments for asthma such as albuterol andSingulair®; agents for treating schizophrenia such as zyprexa,risperdal, seroquel, and haloperidol; anti-inflammatory agents such ascorticosteroids, TNF blockers, IL-1 RA, azathioprine, cyclophosphamide,and sulfasalazine; immunomodulatory and immunosuppressive agents such ascyclosporin, tacrolimus, rapamycin, mycophenolate mofetil, interferons,corticosteroids, cyclophophamide, azathioprine, and sulfasalazine;neurotrophic factors such as acetylcholinesterase inhibitors, MAOinhibitors, interferons, anti-convulsants, ion channel blockers,riluzole, and anti-Parkinsonian agents; agents for treatingcardiovascular disease such as beta-blockers, ACE inhibitors, diuretics,nitrates, calcium channel blockers, and statins, fibrates, cholesterolabsorption inhibitors, bile acid sequestrants, and niacin; agents fortreating liver disease such as corticosteroids, cholestyramine,interferons, and anti-viral agents; agents for treating blood disorderssuch as corticosteroids, anti-leukemic agents, and growth factors;agents for treating immunodeficiency disorders such as gamma globulin;and anti-diabetic agents such as biguanides (metformin, phenformin,buformin), thiazolidinediones (rosiglitazone, pioglitazone,troglitazone), sulfonylureas (tolbutamide, acetohexamide, tolazamide,chlorpropamide, glipizide, glyburide, glimepiride, gliclazide),meglitinides (repaglinide, nateglinide), alpha-glucosidase inhibitors(miglitol, acarbose), incretin mimetics (exenatide, liraglutide,taspoglutide), gastric inhibitory peptide analogs, DPP-4 inhibitors(vildagliptin, sitagliptin, saxagliptin, linagliptin, alogliptin),amylin analogs (pramlintide), and insulin and insulin analogs.

In certain embodiments, compounds of the present invention, or apharmaceutically acceptable composition thereof, are administered incombination with antisense agents, a monoclonal or polyclonal antibodyor an siRNA therapeutic.

Those additional agents may be administered separately from an inventivecompound-containing composition, as part of a multiple dosage regimen.Alternatively or in addition to those additional agents administeredseparately, those agents may be part of a single dosage form, mixedtogether with a compound of this invention in a single composition. Ifadministered as part of a multiple dosage regime, the two active agentsmay be submitted simultaneously, sequentially or within a period of timefrom one another, normally within five hours from one another.

As used herein, the term “combination,” “combined,” and related termsrefers to the simultaneous or sequential administration of therapeuticagents in accordance with this invention. For example, a compound of thepresent invention may be administered with another therapeutic agentsimultaneously or sequentially in separate unit dosage forms or togetherin a single unit dosage form. Accordingly, the present inventionprovides a single unit dosage form comprising a compound of formula I,an additional therapeutic agent, and a pharmaceutically acceptablecarrier, adjuvant, or vehicle.

The amount of both, an inventive compound and additional therapeuticagent (in those compositions which comprise an additional therapeuticagent as described above) that may be combined with the carriermaterials to produce a single dosage form will vary depending upon thehost treated and the particular mode of administration. Preferably,compositions of this invention should be formulated so that a dosage ofbetween 0.01-100 mg/kg body weight/day of an inventive can beadministered.

In those compositions which comprise an additional therapeutic agent,that additional therapeutic agent and the compound of this invention mayact synergistically. Therefore, the amount of additional therapeuticagent in such compositions will be less than that required in amonotherapy utilizing only that therapeutic agent. In such compositionsa dosage of between 0.01-100 μg/kg body weight/day of the additionaltherapeutic agent can be administered.

The amount of additional therapeutic agent present in the compositionsof this invention will be no more than the amount that would normally beadministered in a composition comprising that therapeutic agent as theonly active agent. Preferably the amount of additional therapeutic agentin the presently disclosed compositions will range from about 50% to100% of the amount normally present in a composition comprising thatagent as the only therapeutically active agent.

Therapeutic Entities

Multifunctional agents described herein in many embodiments comprise atleast one therapeutic entity, in addition to a targeting entity (e.g.,compound of formula I) described above. Contemplated therapeuticentities include, without limitation, anti-cancer agents (e.g., agentsthat inhibit tumor growth, agents that inhibit proliferation of cancercells, agents that preferentially kill cancer cells, agents that inhibitangiogenesis, etc.), agents that attenuate any adverse effects (e.g.,antiemetics, etc.) and/or with other approved chemotherapeutic drugs, aswell as adjuvants (e.g., agents that elicit adjuvant effects).

Suitable therapeutic entities include anti-cancer agents can belong toany of various classes of compounds including, but not limited to, smallmolecules, peptides, saccharides, steroids, antibodies, fusion proteins,antisense polynucleotides, ribozymes, small interfering RNAs,peptidomimetics, and the like. Similarly, suitable anti-cancer agentscan be found among any of a variety of classes of anti-cancer agentsincluding, but not limited to, alkylating agents, anti-metabolite drugs,anti-mitotic antibiotics, alkaloidal anti-tumor agents, hormones andanti-hormones, interferons, non-steroidal anti-inflammatory drugs, andvarious other anti-tumor agents.

Examples of chemotherapeutics include, but are not limited to,anti-mitotic agents, alkylating drugs (e.g., mechlorethamine,chlorambucil, cyclophosphamide, melphalan, ifosfamide, etc.),antimetabolites (e.g., methotrexate, etc.), purine antagonists andpyrimidine antagonists (e.g., 6-mercaptopurine, 5-fluorouracil,cytarabine, gemcitabine, etc.), spindle poisons (e.g., vinblastine,vincristine, vinorelbine, paclitaxel, etc.), podophyllotoxins (e.g.,etoposide, irinotecan, topotecan, etc.), antibiotics (e.g., doxorubicin,bleomycin, mitomycin, etc.), nitrosureas (e.g., carmustine, lomustine,nomustine, etc.), inorganic ions (e.g., cisp latin, carboplatin, etc.),enzymes (e.g., asparaginase, etc.), and hormones (e.g., tamoxifen,leuprolide, flutamide, megestrol, etc.), to name a few. For a morecomprehensive discussion of updated cancer therapies see,www.cancer.gov/, a list of the FDA approved oncology drugs athttp://www.fda.gov/cder/cancer/druglistframe.htm, and The Merck Manual,Seventeenth Ed. 1999, the entire contents of which are herebyincorporated by reference.

Non-limiting examples of cytotoxic agents which can be employed as atherapeutic entity for any of the multifunctional agents contemplated inthe present disclosure may be selected from: CHOPP (cyclophosphamide,doxorubicin, vincristine, prednisone, and procarbazine); CHOP(cyclophosphamide, doxorubicin, vincristine, and prednisone); COP(cyclophosphamide, vincristine, and prednisone); CAP-BOP(cyclophosphamide, doxorubicin, procarbazine, bleomycin, vincristine,and prednisone); m-BACOD (methotrexate, bleomycin, doxorubicin,cyclophosphamide, vincristine, dexamethasone, and leucovorin);ProMACE-MOPP (prednisone, methotrexate, doxorubicin, cyclophosphamide,etoposide, leucovorin, mechloethamine, vincristine, prednisone, andprocarbazine); ProMACE-CytaBOM (prednisone, methotrexate, doxorubicin,cyclophosphamide, etoposide, leucovorin, cytarabine, bleomycin, andvincristine); MACOP-B (methotrexate, doxorubicin, cyclophosphamide,vincristine, prednisone, bleomycin, and leucovorin); MOPP(mechloethamine, vincristine, prednisone, and procarbazine); ABVD(adriamycin/doxorubicin, bleomycin, vinblastine, and dacarbazine); MOPP(mechloethamine, vincristine, prednisone and procarbazine) alternatingwith ABV (adriamycin/doxorubicin, bleomycin, and vinblastine); MOPP(mechloethamine, vincristine, prednisone, and procarbazine) alternatingwith ABVD (adriamycin/doxorubicin, bleomycin, vinblastine, anddacarbazine); ChIVPP (chlorambucil, vinblastine, procarbazine, andprednisone); IMVP-16 (ifosfamide, methotrexate, and etoposide); MIME(methyl-gag, ifosfamide, methotrexate, and etoposide); DHAP(dexamethasone, high-dose cytaribine, and cisplatin); ESHAP (etoposide,methylpredisolone, high-dose cytarabine, and cisplatin); CEPP(B)(cyclophosphamide, etoposide, procarbazine, prednisone, and bleomycin);CAMP (lomustine, mitoxantrone, cytarabine, and prednisone); CVP-I(cyclophosphamide, vincristine, and prednisone), ESHOP (etoposide,methylpredisolone, high-dose cytarabine, vincristine and cisplatin);EPOCH (etoposide, vincristine, and doxorubicin for 96 hours with bolusdoses of cyclophosphamide and oral prednisone), ICE (ifosfamide,cyclophosphamide, and etoposide), CEPP(B) (cyclophosphamide, etoposide,procarbazine, prednisone, and bleomycin), CHOP-B (cyclophosphamide,doxorubicin, vincristine, prednisone, and bleomycin), CEPP-B(cyclophosphamide, etoposide, procarbazine, and bleomycin), and P/DOCE(epirubicin or doxorubicin, vincristine, cyclophosphamide, andprednisone).

In some embodiments, chemotherapeutic drugs prescribed for brain tumorsmay be employed as a therapeutic entity in accordance with theinvention. These include, but are not limited to, temozolomide(Temodar®), procarbazine (Matulane), and lomustine (CCNU), which aretaken orally; vincristine (Oncovin® or Vincasar PFS®), cisplatin(Platinol®), carmustine (BCNU, BiCNU), and carboplatin (Paraplatin®),which are administered intravenously; and mexotrexate (Rheumatrex® orTrexall®), which can be administered orally, intravenously orintrathecally (i.e., injected directly into spinal fluid). BCNU is alsogiven under the form of a polymer wafer implant during surgery (Giadel®wafers). One of the most commonly prescribed combination therapy forbrain tumors is PCV (procarbazine, CCNU, and vincristine) which isusually given every six weeks.

In embodiments where the tumor to be treated is a brain tumor ofneuroectodermal origin, a composition or method of the present inventionmay employ agents for the management of symptoms such as seizures andcerebral edema. Examples of anticonvulsants successfully administered tocontrol seizures associated with brain tumors include, but are notlimited to, phenytoin (Dilantin®), Carbamazepine (Tegretol®) anddivalproex sodium (Depakote®). Swelling of the brain may be treated withsteroids (e.g., dexamethasone (Decadron®).

Certain Embodiments of Targeting Moiety Conjugate Agents

In a number of embodiments, the invention provides multifunctionalagents comprising a target entity which essentially consists of acompound of formula I). In such embodiments, therefore, themultifunctional agents according to the present invention are conjugatesof compounds of formula I. Non-limiting embodiments of useful conjugatesare provided below.

For example, provided conjugates comprise a compound of formula I and anucleic acid molecule that is useful as a therapeutic (e.g.,anti-cancer) agent. A variety of chemical types and structural forms ofnucleic acid can be suitable for such strategies. These include, by wayof non-limiting example, DNA, including single-stranded (ssDNA) anddouble-stranded (dsDNA); RNA, including, but not limited to ssRNA,dsRNA, tRNA, mRNA, rRNA, enzymatic RNA; RNA:DNA hybrids, triplexed DNA(e.g., dsDNA in association with a short oligonucleotide), and the like.

In some embodiments, the nucleic acid agent is between about 5 and 2000nucleotides long. In some embodiments, the nucleic acid agent is atleast about 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21,22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39,40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50 or more nucleotides long. Insome embodiments, the nucleic acid agent is less than about 2000, 1900,1800, 1700, 1600, 1500, 1400, 1300, 1200, 1100, 1000, 900, 800, 700,600, 500, 450, 400, 350, 300, 250, 200, 150, 100, 50, 45, 40, 35, 30,25, 20 or fewer nucleotides long.

In some embodiments, the nucleic acid agent comprises a promoter and/orother sequences that regulate transcription. In some embodiments, thenucleic acid agent comprises an origin of replication and/or othersequences that regulate replication. In some embodiments, the nucleicacid agent does not include a promoter and/or an origin of replication.

Nucleic acid anti-cancer agents suitable for use in the practice of thepresent invention include those agents that target genes associated withtumorigenesis and cell growth or cell transformation (e.g.,proto-oncogenes, which code for proteins that stimulate cell division),angiogenic/anti-angiogenic genes, tumor suppressor genes (which code forproteins that suppress cell division), genes encoding proteinsassociated with tumor growth and/or tumor migration, and suicide genes(which induce apoptosis or other forms of cell death), especiallysuicide genes that are most active in rapidly dividing cells.

Examples of genes associated with tumorigenesis and/or celltransformation include MLL fusion genes, BCR-ABL, TEL-AML1, EWS-FLI1,TLS-FUS, PAX3-FKHR, Bcl-2, AML1-ETO, AML1-MTG8, Ras, Fos PDGF, RET, APC,NF-1, Rb, p53, MDM2 and the like; overexpressed genes such as multidrugresistance genes; cyclins; beta-Catenin; telomerase genes; c-myc, n-myc,Bcl-2, Erb-B1 and Erb-B2; and mutated genes such as Ras, Mos, Raf, andMet. Examples of tumor suppressor genes include, but are not limited to,p53, p21, RB1, WT1, NF1, VHL, APC, DAP kinase, p16, ARF, Neurofibromin,and PTEN. Examples of genes that can be targeted by nucleic acid agentsuseful in anti-cancer therapy include genes encoding proteins associatedwith tumor migration such as integrins, selectins, andmetalloproteinases; anti-angiogenic genes encoding proteins that promoteformation of new vessels such as Vascular Endothelial Growth Factor(VEGF) or VEGFr; anti-angiogenic genes encoding proteins that inhibitneovascularization such as endostatin, angiostatin, and VEGF-R2; andgenes encoding proteins such as interleukins, interferon, fibroblastgrowth factor (α-FGF and (β-FGF), insulin-like growth factor (e.g.,IGF-1 and IGF-2), Platelet-derived growth factor (PDGF), tumor necrosisfactor (TNF), Transforming Growth Factor (e.g., TGF-α and TGF-β,Epidermal growth factor (EGF), Keratinocyte Growth Factor (KGF), stemcell factor and its receptor c-Kit (SCF/c-Kit) ligand, CD40L/CD40, VLA-4VCAM-1, ICAM-1/LFA-1, hyalurin/CD44, and the like. As will be recognizedby one skilled in the art, the foregoing examples are not exclusive.

Nucleic acid agents suitable for use in the invention may have any of avariety of uses including, for example, use as anti-cancer or othertherapeutic agents, probes, primers, etc. Nucleic acid agents may haveenzymatic activity (e.g., ribozyme activity), gene expression inhibitoryactivity (e.g., as antisense or siRNA agents, etc), and/or otheractivities. Nucleic acids agents may be active themselves or may bevectors that deliver active nucleic acid agents (e.g., throughreplication and/or transcription of a delivered nucleic acid). Forpurposes of the present specification, such vector nucleic acids areconsidered “therapeutic agents” if they encode or otherwise deliver atherapeutically active agent, even if they do not themselves havetherapeutic activity.

In certain embodiments, conjugates comprise a nucleic acid therapeuticagent that comprises or encodes an antisense compound. The terms“antisense compound or agent,” “antisense oligomer,” “antisenseoligonucleotide,” and “antisense oligonucleotide analog” are used hereininterchangeably, and refer to a sequence of nucleotide bases and asubunit-to-subunit backbone that allows the antisense compound tohybridize to a target sequence in an RNA by Watson-Crick base pairing toform an RNA oligomer heteroduplex within the target sequence. Theoligomer may have exact sequence complementarity within the targetsequence or near complementarity. Such antisense oligomers may block orinhibit translation of the mRNA containing the target sequence, orinhibit gene transcription. Antisense oligomers may bind todouble-stranded or single-stranded sequences.

Examples of antisense oligonucleotides suitable for use in the practiceof the present invention include, for example, those mentioned in thefollowing reviews: R. A Stahel et al., Lung Cancer, 2003, 41: S81-S88;K. F. Pirollo et al., Pharmacol. Ther., 2003, 99: 55-77; A. C. Stephensand R. P. Rivers, Curr. Opin. Mol. Ther., 2003, 5: 118-122; N. M. Deanand C. F. Bennett, Oncogene, 2003, 22: 9087-9096; N. Schiavone et al.,Curr. Pharm. Des., 2004, 10: 769-784; L. Vidal et al., Eur. J. Cancer,2005, 41: 2812-2818; T. Aboul-Fadl, Curr. Med. Chem., 2005, 12:2193-2214; M. E. Gleave and B. P. Monia, Nat. Rev. Cancer, 2005, 5:468-479; Y. S. Cho-Chung, Curr. Pharm. Des., 2005, 11: 2811-2823; E.Rayburn et al., Lett. Drug Design & Discov., 2005, 2: 1-18; E. R.Rayburn et al., Expert Opin. Emerg. Drugs, 2006, 11: 337-352; I. Tammand M. Wagner, Mol. Biotechnol., 2006, 33: 221-238 (each of which isincorporated herein by reference in its entirety).

Examples of suitable antisense oligonucleotides include, for exampleoblimersen sodium (also known as Genasense™ or G31239, developed byGenta, Inc., Berkeley Heights, N.J.), a phosphorothioate oligomertargeted towards the initiation codon region of the bcl-2 mRNA. Bcl-2 isa potent inhibitor of apoptosis and is overexpressed in many cancerincluding follicular lymphomas, breast cancer, colon cancer, prostatecancer, and intermediate/high-grade lymphomas (C. A. Stein et al.,Semin. Oncol., 2005, 32: 563-573; S. R. Frankel, Semin. Oncol., 2003,30: 300-304). Other suitable antisense oligonucleotides include GEM-231(HYB0165, Hybridon, Inc., Cambridge, Mass.), which is a mixed backboneoligonucleotide directed against cAMP-dependent protein kinase A (PKA)(S. Goel et al., Clin. Cancer Res., 203, 9: 4069-4076); Affinitak (ISIS3521 or aprinocarsen, ISIS pharmaceuticals, Inc., Carlsbad, Calif.), anantisense inhibitor of PKCalpha; OGX-011 (Isis 112989, IsisPharmaceuticals, Inc.), a 2′-methoxyethyl modified antisenseoligonucleotide against clusterin, a glycoprotein implicated in theregulation of the cell cycle, tissue remodeling, lipid transport, andcell death and which is overexpressed in cancers of breast, prostate andcolon; ISIS 5132 (Isis 112989, Isis Pharmaceuticals, Inc.), aphosphorothioate oligonucleotide complementary to a sequence of the3′-unstranslated region of the c-raf-1 mRNA (S. P. Henry et al.,Anticancer Drug Des., 1997, 12: 409-420; B. P. Monia et al., Proc. Natl.Acad. Sci. USA, 1996, 93: 15481-15484; C. M. Rudin et al., Clin. CancerRes., 2001, 7: 1214-1220); ISIS 2503 (Isis Pharmaceuticals, Inc.), aphosphorothioate oligonucleotide antisense inhibitor of human H-ras mRNAexpression (J. Kurreck, Eur. J. Biochem., 2003, 270: 1628-1644);oligonucleotides targeting the X-linked inhibitor of apoptosis protein(XIAP), which blocks a substantial portion of the apoptosis pathway,such as GEM 640 (AEG 35156, Aegera Therapeutics Inc. and Hybridon, Inc.)or targeting survivin, an inhibitor of apoptosis protein (IAP), such asISIS 23722 (Isis Pharmaceuticals, Inc.), a 2′-O-methoxyethyl chimericoligonucleotide; MG98, which targets DNA methyl transferase; andGTI-2040 (Lorus Therapeutics, Inc. Toronto, Canada), a 20-meroligonucleotide that is complementary to a coding region in the mRNA ofthe R2 small subunit component of human ribonucleotide reductase.

Other suitable antisense oligonucleotides include antisenseoligonucleotides that are being developed against Her-2/neu, c-Myb,c-Myc, and c-Raf (see, for example, A. Biroccio et al., Oncogene, 2003,22: 6579-6588; Y. Lee et al., Cancer Res., 2003, 63: 2802-2811; B. Lu etal., Cancer Res., 2004, 64: 2840-2845; K. F. Pirollo et al., Pharmacol.Ther., 2003, 99: 55-77; and A. Rait et al., Ann. N. Y. Acad. Sci., 2003,1002: 78-89).

In certain embodiments, conjugates of the present invention comprise anucleic acid anti-cancer agent that comprises or encodes an interferingRNA molecule. The terms “interfering RNA” and “interfering RNA molecule”are used herein interchangeably, and refer to an RNA molecule that caninhibit or downregulate gene expression or silence a gene in asequence-specific manner, for example by mediating RNA interference(RNAi). RNA interference (RNAi) is an evolutionarily conserved,sequence-specific mechanism triggered by double-stranded RNA (dsRNA)that induces degradation of complementary target single-stranded mRNAand “silencing” of the corresponding translated sequences (McManus andSharp, 2002, Nature Rev. Genet., 2002, 3: 737). RNAi functions byenzymatic cleavage of longer dsRNA strands into biologically active“short-interfering RNA” (siRNA) sequences of about 21-23 nucleotides inlength (Elbashir et al., Genes Dev., 2001, 15: 188). RNA interferencehas emerged as a promising approach for therapy of cancer.

An interfering RNA suitable for use in the practice of the presentinvention can be provided in any of several forms. For example, aninterfering RNA can be provided as one or more of an isolated shortinterfering RNA (siRNA), double-stranded RNA (dsRNA), micro-RNA (miRNA),or short hairpin RNA (shRNA).

Examples of interfering RNA molecules suitable for use in the presentinvention include, for example, the iRNAs cited in the followingreviews: O. Milhavet et al., Pharmacol. Rev., 2003, 55: 629-648; F. Biet al., Curr. Gene. Ther., 2003, 3: 411-417; P. Y. Lu et al., Curr.Opin. Mol. Ther., 2003, 5: 225-234; I. Friedrich et al., Semin. CancerBiol., 2004, 14: 223-230; M. Izquierdo, Cancer Gene Ther., 2005, 12:217-227; P. Y. Lu et al., Adv. Genet., 2005, 54: 117-142; G. R. Devi,Cancer Gene Ther., 2006, 13: 819-829; M. A. Behlke, Mol. Ther., 2006,13: 644-670; and L. N. Putral et al., Drug News Perspect., 2006, 19:317-324 (the contents of each of which are incorporated herein byreference in their entirety).

Other examples of suitable interfering RNA molecules include, but arenot limited to, p53 interfering RNAs (e.g., T. R. Brummelkamp et al.,Science, 2002, 296: 550-553; M. T. Hemman et al., Nat. Genet., 2003, 33:396-400); interfering RNAs that target the bcr-abl fusion, which isassociated with development of chronic myeloid leukemia and acutelymphoblastic leukemia (e.g., M. Scherr et al., Blood, 2003, 101:1566-1569; M. J. Li et al., Oligonucleotides, 2003, 13: 401-409),interfering RNAs that inhibit expression of NPM-ALK, a protein that isfound in 75% of anaplastic large cell lymphomas and leads to expressionof a constitutively active kinase associated with tumor formation (U.Ritter et al., Oligonucleotides, 2003, 13: 365-373); interfering RNAsthat target oncogenes, such as Raf-1 (T. F. Lou et al.,Oligonucleotides, 2003, 13: 313-324), K-Ras (T. R. Brummelkamp et al.,Cancer Cell, 2002, 2: 243-247), erbB-2 (G. Yang et al., J. Biol. Chem.,2004, 279: 4339-4345); interfering RNAs that target b-catenin protein,whose over-expression leads to transactivation of the T-cell factortarget genes, which is thought to be the main transforming event incolorectal cancer (M. van de Wetering et al., EMBO Rep., 2003, 4:609-615).

In certain embodiments, conjugates of the present invention comprise anucleic acid therapeutic agent that is a ribozyme. As used herein, theterm “ribozyme” refers to a catalytic RNA molecule that can cleave otherRNA molecules in a target-specific marmer Ribozymes can be used todownregulate the expression of any undesirable products of genes ofinterest. Examples of ribozymes that can be used in the practice of thepresent invention include, but are not limited to, ANGIOZYME™ (RPI.4610,Sima Therapeutics, Boulder, Colo.), a ribozyme targeting the conservedregion of human, mouse, and rat vascular endothelial growth factorreceptor (VEGFR)-1 mRNA, and Herzyme (Sima Therapeutics).

In certain embodiments, entities or moieties within conjugates of theinvention comprise a photosensitizer used in photodynamic therapy (PDT).In PDT, local or systemic administration of a photosensitizer to apatient is followed by irradiation with light that is absorbed by thephotosensitizer in the tissue or organ to be treated. Light absorptionby the photosensitizer generates reactive species (e.g., radicals) thatare detrimental to cells. For maximal efficacy, a photosensitizertypically is in a form suitable for administration, and also in a formthat can readily undergo cellular internalization at the target site,often with some degree of selectivity over normal tissues.

While some photosensitizers (e.g., Photofrin®, QLT, Inc., Vancouver, BC,Canada) have been delivered successfully as part of a simple aqueoussolution, such aqueous solutions may not be suitable for hydrophobicphotosensitizer drugs, such as those that have a tetra- orpoly-pyrrole-based structure. These drugs have an inherent tendency toaggregate by molecular stacking, which results in a significantreduction in the efficacy of the photosensitization processes (Siggel etal., J. Phys. Chem., 1996, 100: 2070-2075). Approaches to minimizeaggregation include liposomal formulations (e.g., for benzoporphyrinderivative monoacid A, BPDMA, Verteporfin®, QLT, Inc., Vancouver,Canada; and zinc phthalocyanine, CIBA-Geigy, Ltd., Basel, Switzerland),and conjugation of photosensitizers to biocompatible block copolymers(Peterson et al., Cancer Res., 1996, 56: 3980-3985) and/or antibodies(Omelyanenko et al., Int. J. Cancer, 1998, 75: 600-608).

Conjugates of the invention comprising a compound of the inventionassociated with a photosensitizer can be used as new delivery systems inPDT. In addition to reducing photosensitizer aggregation, delivery ofphotosensitizers according to the present invention exhibits otheradvantages such as increased specificity for target tissues/organ andcellular internalization of the photosensitizer.

Photosensitizers suitable for use in the present invention include anyof a variety of synthetic and naturally occurring molecules that havephotosensitizing properties useful in PDT. In certain embodiments, theabsorption spectrum of the photosensitizer is in the visible range,typically between 350 nm and 1200 nm, preferably between 400 nm and 900nm, e.g., between 600 nm and 900 nm. Suitable photosensitizers that canbe coupled to toxins according to the present invention include, but arenot limited to, porphyrins and porphyrin derivatives (e.g., chlorins,bacteriochlorins, isobacteriochlorins, phthalocyanines, andnaphthalocyanines); metalloporphyrins, metallophthalocyanines,angelicins, chalcogenapyrrillium dyes, chlorophylls, coumarins, flavinsand related compounds such as alloxazine and riboflavin, fullerenes,pheophorbides, pyropheophorbides, cyanines (e.g., merocyanine 540),pheophytins, sapphyrins, texaphyrins, purpurins, porphycenes,phenothiaziniums, methylene blue derivatives, naphthalimides, nile bluederivatives, quinones, perylenequinones (e.g., hypericins, hypocrellins,and cercosporins), psoralens, quinones, retinoids, rhodamines,thiophenes, verdins, xanthene dyes (e.g., eosins, erythrosins, rosebengals), dimeric and oligomeric forms of porphyrins, and prodrugs suchas 5-aminolevulinic acid (R. W. Redmond and J. N. Gamlin, Photochem.Photobiol., 1999, 70: 391-475).

Exemplary photosensitizers suitable for use in the present inventioninclude those described in U.S. Pat. Nos. 5,171,741; 5,171,749;5,173,504; 5,308,608; 5,405,957; 5,512,675; 5,726,304; 5,831,088;5,929,105; and 5,880,145 (the contents of each of which are incorporatedherein by reference in their entirety).

In certain embodiments, conjugates of the invention comprise aradiosensitizer. As used herein, the term “radiosensitizer” refers to amolecule, compound or agent that makes tumor cells more sensitive toradiation therapy. Administration of a radiosensitizer to a patientreceiving radiation therapy generally results in enhancement of theeffects of radiation therapy. Ideally, a radiosensitizer exerts itsfunction only on target cells. For ease of use, a radiosensitizer shouldalso be able to find target cells even if it is administeredsystemically. However, currently available radiosensitizers aretypically not selective for tumors, and they are distributed bydiffusion in a mammalian body. Conjugates of the present invention canbe used as a new delivery system for radiosensitizers.

A variety of radiosensitizers are known in the art. Examples ofradiosensitizers suitable for use in the present invention include, butare not limited to, paclitaxel (TAXOL), carboplatin, cisplatin, andoxaliplatin (Amorino et al., Radiat. Oncol. Investig. 1999; 7: 343-352;Choy, Oncology, 1999, 13: 22-38; Safran et al., Cancer Invest., 2001,19: 1-7; Dionet et al., Anticancer Res., 2002, 22: 721-725; Cividalli etal., Radiat. Oncol. Biol. Phys., 2002, 52: 1092-1098); gemcitabine(Gemzar®) (Choy, Oncology, 2000, 14: 7-14; Mornex and Girard, Annals ofOncology, 2006, 17: 1743-1747); etanidazole (Nitrolmidazole®) (Inanamiet al., Int. J. Radiat. Biol., 2002, 78: 267-274); misonidazole(Tamulevicius et al., Br. J. Radiology, 1981, 54: 318-324; Palcic etal., Radiat. Res., 1984, 100: 340-347), tirapazamine (Masunaga et al.,Br. J. Radiol., 2006, 79: 991-998; Rischin et al., J. Clin. Oncol.,2001, 19: 535-542; Shulman et al., Int. J. Radiat. Oncol. Biol. Phys.,1999, 44: 349-353); and nucleic acid base derivatives, e.g., halogenatedpurines or pyrimidines, such as 5-fluorodeoxyuridine (Buchholz et al.,Int. J. Radiat. Oncol. Biol. Phys., 1995, 32: 1053-1058).

In certain embodiments, conjugates of the invention comprise aradioisotope. Examples of suitable radioisotopes include any α-, β- orγ-emitter, which, when localized at a tumor site, results in celldestruction (S. E. Order, “Analysis, Results, and Future Prospective ofthe Therapeutic Use of Radiolabeled Antibody in Cancer Therapy”,Monoclonal Antibodies for Cancer Detection and Therapy, R. W. Baldwin etal. (Eds.), Academic Press, 1985). Examples of such radioisotopesinclude, but are not limited to, iodine-131 (¹³¹I), iodine-125 (¹²⁵I),bismuth-212 (²¹²Bi), bismuth-213 (²¹³Bi), astatine-211 (²¹¹At),rhenium-186 (¹⁸⁶Re), rhenium-188 (¹⁸⁸Re), phosphorus-32 (³²P),yttrium-90 (⁹⁰yY), samarium-153 (¹⁵³Sm), and lutetium-177 (¹⁷⁷Lu).

In certain embodiments, conjugates of the invention comprise asuperantigen or biologically active portion thereof. Superantigensconstitute a group of bacterial and viral proteins that are extremelyefficient in activating a large fraction of the T-cell population.Superantigens bind directly to the major histocompatibility complex(MHC) without being processed. In fact, superantigens bind unprocessedoutside the antigenbinding groove on the MHC class II molecules, therebyavoiding most of the polymorphism in the conventional peptide-bindingsite.

A superantigen-based tumor therapeutic approach has been developed forthe treatment of solid tumors. In this approach, a targeting moiety, forexample, an antibody or antibody fragment, is conjugated to asuperantigen, providing a targeted superantigen. If the antibody, orantibody fragment, recognizes a tumor-associated antigen, the targetedsuperantigen, bound to tumors cells, can trigger superantigen-activatedcytotoxic T-cells to kill the tumor cells directly bysuperantigen-dependent cell mediated cytotoxicity. (See, e.g., Søgaardet al., (1996) “Antibody-targeted superantigens in cancerimmunotherapy,” Immunotechnology, 2(3): 151-162, the entire contents ofwhich are herein incorporated by reference.)

Superantigen-based tumor therapeutics have had some success. Forexample, fusion proteins with wild-type staphylococcal enterotoxin A(SEA) have been investigated in clinical trials of colorectal andpancreatic cancer (Giantonio et al., J. Clin. Oncol., 1997, 15:1994-2007; Alpaugh et a., Clin. Cancer Res., 1998, 4: 1903-1914; Chenget al., J. Clin. Oncol., 2004, 22: 602-609; the entire contents of eachof which are herein incorporated by reference); staphylococcalsuperantigens of the enterotoxin gene cluster (egc) have been studiedfor the treatment of non-small cell lung cancer (Terman et al., Clin.Chest Med., 2006, 27: 321-324, the entire contents of which are hereinincorporated by reference), and staphylococcal enterotoxin B has beenevaluated for the intravesical immunotherapy of superficial bladdercancer (Perabo et al., Int. J. Cancer, 2005, 115: 591-598, the entirecontents of which are herein incorporated by reference).

A superantigen, or a biologically active portion thereof, can beassociated to a compound of the invention to form a conjugate accordingto the present invention and used in a therapy, e.g., an anti-cancertherapy, as described herein.

Examples of superantigens suitable for use in the present inventioninclude, but are not limited to, staphylococcal enterotoxin (SE) (e.g.,staphylococcal enterotoxin A (SEA) or staphylococcal enterotoxin E(SEE)), Streptococcus pyogenes exotoxin (SPE), Staphylococcus aureustoxic shock-syndrome toxin (TSST-1), streptococcal mitogenic exotoxin(SME), streptococcal superantigen (SSA), and staphylococcalsuperantigens of the enterotoxin gene cluster. As known to one skilledin the art, the three-dimensional structures of the above listedsuperantigens can be obtained from the Protein Data Bank. Similarly, thenucleic acid sequences and the amino acid sequences of the above listedsuperantigens and other superantigens can be obtained from GenBank.

In certain embodiments, a conjugate of the present invention may be usedin directed enzyme prodrug therapy. In a directed enzyme prodrug therapyapproach, a directed/targeted enzyme and a prodrug are administered to asubject, wherein the targeted enzyme is specifically localized to aportion of the subject's body where it converts the prodrug into anactive drug. The prodrug can be converted to an active drug in one step(by the targeted enzyme) or in more than one step. For example, theprodrug can be converted to a precursor of an active drug by thetargeted enzyme. The precursor can then be converted into the activedrug by, for example, the catalytic activity of one or more additionaltargeted enzymes, one or more non-targeted enzymes administered to thesubject, one or more enzymes naturally present in the subject or at thetarget site in the subject (e.g., a protease, phosphatase, kinase orpolymerase), by an agent that is administered to the subject, and/or bya chemical process that is not enzymatically catalyzed (e.g., oxidation,hydrolysis, isomerization, epimerization, etc.).

Different approaches have been used to direct/target the enzyme to thesite of interest. For example, in ADEPT (antibody-directed enzymeprodrug therapy), an antibody designed/developed against a tumor antigenis linked to an enzyme and injected in a subject, resulting in selectivebinding of the enzyme to the tumor. When the discrimination betweentumor and normal tissue enzyme levels is sufficient, a prodrug isadministered to the subject. The prodrug is converted to its active formby the enzyme only within the tumor. Selectivity is achieved by thetumor specificity of the antibody and by delaying prodrug administrationuntil there is a large differential between tumor and normal tissueenzyme levels. Early clinical trials are promising and indicate thatADEPT may become an effective treatment for all solid cancers for whichtumor-associated or tumor-specific antibodies are known. Tumors havealso been targeted with the genes encoding for prodrug activatingenzymes. This approach has been called virus-directed enzyme prodrugtherapy (VDEPT) or more generally GDEPT (gene-directed enzyme prodrugtherapy, and has shown good results in laboratory systems. Otherversions of directed enzyme prodrug therapy include PDEPT(polymer-directed enzyme prodrug therapy), LEAPT (lectin-directedenzyme-activated prodrug therapy), and CDEPT (clostridial-directedenzyme prodrug therapy). A conjugate according to the present invention,which comprises a prodrug activating enzyme associated with a compoundof the invention, can be used in a similar way.

Nonlimiting examples of enzyme/prodrug/active drug combinations suitablefor use in the present invention are described, for example, in Bagshaweet al., Current Opinions in Immunology, 1999, 11: 579-583; Wilman,“Prodrugs in Cancer Therapy”, Biochemical Society Transactions, 14:375-382, 615^(th) Meeting, Belfast, 1986; Stella et al., “Prodrugs: AChemical Approach To Targeted Drug Delivery”, in “Directed DrugDelivery”, Borchardt et al., (Eds), pp. 247-267 (Humana Press, 1985).Nonlimiting examples of enzyme/prodrug/active anti-cancer drugcombinations are described, for example, in Rooseboom et al., Pharmacol.Reviews, 2004, 56: 53-102.

Examples of prodrug activating enzymes include, but are not limited to,nitroreductase, cytochrome P450, purine-nucleoside phosphorylase,thymidine kinase, alkaline phosphatase, β-glucuronidase,carboxypeptidase, penicillin amidase, β-lactamase, cytosine deaminase,and methionine γ-lyase.

Examples of anti-cancer drugs that can be formed in vivo by activationof a prodrug by a prodrug activating enzyme include, but are not limitedto, 5-(aziridin-1-yl)-4-hydroxyl-amino-2-nitro-benzamide,isophosphoramide mustard, phosphoramide mustard, 2-fluoroadenine,6-methylpurine, ganciclovir-triphosphate nucleotide, etoposide,mitomycin C, p-[N,N-bis(2-chloroethyl)amino]phenol (POM), doxorubicin,oxazolidinone, 9-aminocamptothecin, mustard, methotrexate, benzoic acidmustard, doxorubicin, adriamycin, daunomycin, carminomycin, bleomycins,esperamicins, melphalan, palytoxin, 4-desacetylvinblastine-3-carboxylicacid hydrazide, phenylenediamine mustard,4′-carboxyphthalato(1,2-cyclohexane-diamine) platinum, taxol,5-fluorouracil, methylselenol, and carbonothionic difluoride.

In certain embodiments, a therapeutic (e.g., anti-cancer) agent within aconjugate of the present invention comprises an anti-angiogenic agent.Antiangiogenic agents suitable for use in the present invention includeany molecule, compound, or factor that blocks, inhibits, slows down, orreduces the process of angiogenesis, or the process by which new bloodvessels form by developing from preexisting vessels. Such a molecule,compound, or factor can block angiogenesis by blocking, inhibiting,slowing down, or reducing any of the steps involved in angiogenesis,including (but not limited to) steps of (1) dissolution of the membraneof the originating vessel, (2) migration and proliferation ofendothelial cells, and (3) formation of new vasculature by migratingcells.

Examples of anti-angiogenic agents include, but are not limited to,bevacizumab (AVASTIN®), celecoxib (CELEBREX®), endostatin, thalidomide,EMD121974 (Cilengitide), TNP-470, squalamine, combretastatin A4,interferon-α, anti-VEGF antibody, SU5416, SU6668, PTK787/2K 22584,Marimistal, AG3340, COL-3, Neovastat, and BMS-275291.

Anti-angiogenic agents may be used in a variety of therapeutic contexts,including, but not limited to, anti-cancer therapies and therapies formacular degeneration.

As will be recognized by one skilled in the art, the specific examplesof therapeutic agents cited herein represent only a very small number ofthe therapeutic agents that are suitable for use in the practice of thepresent invention.

Detection Entities

Multifunctional agents described herein in many embodiments comprise atleast one detection entity, in addition to a targeting entity describedabove.

A detection entity may be any entity that allows detection of atargeting agent after binding to a tissue or localization at a system ofinterest. Any of a wide variety of detectable agents can be used asdetection entity (e.g., labeling moieties) in multifunctional conjugateagents of the present invention. A detection entity may be directlydetectable or indirectly detectable. Examples of detection entityinclude, but are not limited to: various ligands, radionuclides (e.g.,³H, ¹⁴C, ¹⁸F, ¹⁹F, ³²P, ³⁵S, ¹³⁵I, ¹²⁵I, ¹²³I, ⁶⁴Cu, ¹⁸⁷Re, ¹¹¹In, ⁹⁰Y,^(99m)Tc, ¹⁷⁷Lu, etc.), fluorescent dyes (for specific exemplaryfluorescent dyes, see below), chemiluminescent agents (such as, forexample, acridinum esters, stabilized dioxetanes, and the like),bioluminescent agents, spectrally resolvable inorganic fluorescentsemiconductors nanocrystals (i.e., quantum dots), metal nanoparticles(e.g., gold, silver, copper, platinum, etc.) nanoclusters, paramagneticmetal ions, enzymes (for specific examples of enzymes, see below),colorimetric labels (such as, for example, dyes, colloidal gold, and thelike), biotin, dioxigenin, haptens, and proteins for which antisera ormonoclonal antibodies are available.

In certain embodiments, a detection entity comprises a fluorescentlabel. Numerous known fluorescent labeling moieties of a wide variety ofchemical structures and physical characteristics are suitable for use inthe practice of methods of diagnosis of the present invention. Suitablefluorescent dyes include, but are not limited to, fluorescein andfluorescein dyes (e.g., fluorescein isothiocyanine or FITC,naphthofluorescein, 4′,5′-dichloro-2′,7′-dimethoxyfluorescein,carboxyfluorescein or FAM, etc.), carbocyanine, merocyanine, styryldyes, oxonol dyes, phycoerythrin, erythrosin, eosin, rhodamine dyes(e.g., carboxytetramethyl-rhodamine or TAMRA, carboxyrhodamine 6G,carboxy-X-rhodamine (ROX), lissamine rhodamine B, rhodamine 6G,rhodamine Green, rhodamine Red, tetramethylrhodamine (TMR), etc.),coumarin and coumarin dyes (e.g., methoxycoumarin, dialkylaminocoumarin,hydroxycoumarin, aminomethylcoumarin (AMCA), etc.), Oregon Green Dyes(e.g., Oregon Green 488, Oregon Green 500, Oregon Green 514., etc.),Texas Red, Texas Red-X, Spectrum Red™, Spectrum Green™, cyanine dyes(e.g., Cy-3™, Cy-5™, Cy-3.5™, Cy-5.5™ etc.), Alexa Fluor dyes (e.g.,Alexa Fluor 350, Alexa Fluor 488, Alexa Fluor 532, Alexa Fluor 546,Alexa Fluor 568, Alexa Fluor 594, Alexa Fluor 633, Alexa Fluor 660,Alexa Fluor 680, etc.), BODIPY dyes (e.g., BODIPY FL, BODIPY R6G, BODIPYTMR, BODIPY TR, BODIPY 530/550, BODIPY 558/568, BODIPY 564/570, BODIPY576/589, BODIPY 581/591, BODIPY 630/650, BODIPY 650/665, etc.), IRDyes(e.g., IRD40, IRD 700, IRD 800, etc.), and the like. For more examplesof suitable fluorescent dyes and methods for coupling fluorescent dyesto other chemical entities such as proteins and peptides, see, forexample, “The Handbook of Fluorescent Probes and Research Products”,9¹¹¹Ed., Molecular Probes, Inc., Eugene, Oreg.

Favorable properties of fluorescent labeling agents include high molarabsorption coefficient, high fluorescence quantum yield, andphotostability. In certain embodiments, labeling fluorophores desirablyexhibit absorption and emission wavelengths in the visible (i.e.,between 400 and 750 nm) rather than in the ultraviolet range of thespectrum (i.e., lower than 400 nm).

In certain embodiments, a detection entity comprises an enzyme. Examplesof suitable enzymes include, but are not limited to, those used in anELISA, e.g., horseradish peroxidase, beta-galactosidase, luciferase,alkaline phosphatase, etc. Other examples include beta-glucuronidase,beta-D-glucosidase, urease, glucose oxidase, etc. An enzyme may beconjugated to a targeting entity (e.g., compound of the invention offormula I) using a linker group such as a carbodiimide, a diisocyanate,a glutaraldehyde, and the like. More detailed description of suitablelinkers is provided elsewhere herein.

In certain embodiments, a detection entity comprises a radioisotope thatis detectable by Single Photon Emission Computed Tomography (SPECT) orPosition Emission Tomography (PET). Examples of such radionuclidesinclude, but are not limited to, iodine-131 (¹³¹I), iodine-125 (¹²⁵I),bismuth-212 (²¹²Bi), bismuth-213 (²¹³Bi), astatine-221 (²¹¹At),copper-67 (⁶⁷Cu), copper-64 (⁶⁴Cu), rhenium-186 (¹⁸⁶Re), rhenium-186(¹⁸⁸Re), phosphorus-32 (³²P), samarium-153 (¹⁵³Sm), lutetium-177 (¹¹⁷Lu)technetium-99m (^(99m)Tc), gallium-67 (⁶⁷Ga), indium-I11 (¹¹¹In), andthallium-201 (²⁰¹Tl).

In certain embodiments, a labeling moiety comprises a radioisotope thatis detectable by Gamma camera. Examples of such radioisotopes include,but are not limited to, iodine-131 (¹³¹I), and technetium-99m(^(99m)Tc).

In certain embodiments, a detection entity comprises a paramagneticmetal ion that is a good contrast enhancer in Magnetic Resonance Imaging(MRI). Examples of such paramagnetic metal ions include, but are notlimited to, gadolinium III (Gd³⁺), chromium III (Cr³⁺), dysprosium III(Dy³⁺), iron III (Fe³⁺), manganese II (Mn²⁺), and ytterbium III (Yb³⁺).In certain embodiments, the detection entity comprises gadolinium III(Gd³⁺). Gadolinium is an FDA-approved contrast agent for MRI, whichaccumulates in abnormal tissues causing these abnormal areas to becomevery bright (enhanced) on the magnetic resonance image. Gadolinium isknown to provide great contrast between normal and abnormal tissues indifferent areas of the body, in particular in the brain.

In certain embodiments, a labeling moiety comprises a stableparamagnetic isotope detectable by nuclear magnetic resonancespectroscopy (MRS). Examples of suitable stable paramagnetic isotopesinclude, but are not limited to, carbon-13 (¹³C) and fluorine-19 (¹⁹F).

Conjugation

As stated above, multifunctional agents described herein comprisemultiple entities, each having at least one function. As already noted,certain embodiments of contemplated multifunctional agents comprise atargeting entity and at least one of the following entities: a detectionentity and a therapeutic entity. In some embodiments, a multifunctionalagent of the invention contains a targeting entity and a therapeuticentity; but not a detection entity. In some embodiments, amultifunctional agent of the invention contains a targeting entity; adetection entity; but not a therapeutic entity. In some embodiments, amultifunctional agent of the invention contains a targeting entity; atherapeutic entity; and a detection entity. In any of contemplatedembodiments, the entities of an agent are conjugated to one another.Conjugation of various entities to form a multifunctional agent is notlimited to particular modes of conjugation. For example, two entitiesmay be covalently conjugated directly to each other. Alternatively, twoentities may be indirectly conjugated to each other, such as via alinker entity. In some embodiments, a multifunctional agent may includedifferent types of conjugation within the agent, such that some entitiesof the agent are conjugated via direct conjugation while other entitiesof the agent are indirectly conjugated via one or more linkers. In someembodiments, a multifunctional agent of the invention comprises a singletype of a linker entity. In other embodiments, a multifunctional agentof the invention comprises more than one types of a linker entities. Insome embodiments, a multifunctional agent includes a single type oflinker entities but of varying length.

In many of the embodiments described herein, association between oramongst entities contained in a multifunctional agent is covalent. Aswill be appreciated by one skilled in the art, the moieties may beattached to each other either directly or indirectly (e.g., through alinker, as described above).

In certain embodiments, where one entity (such as a targeting entity)and a second entity of a multifunctional agent are directly covalentlylinked to each other, such direct covalent conjugation can be through alinkage (e.g., a linker or linking entity) such as an amide, ester,carbon-carbon, disulfide, carbamate, ether, thioether, urea, amine, orcarbonate linkage. Covalent conjugation can be achieved by takingadvantage of functional groups present on the first entity and/or thesecond entity of the multifunctional agent. Alternatively, anon-critical amino acid may be replaced by another amino acid that willintroduce a useful group (such as amino, carboxy or sulfhydryl) forcoupling purposes. Alternatively, an additional amino acid may be addedto at least one of the entities of the multifunctional agent tointroduce a useful group (such as amino, carboxy or sulfhydryl) forcoupling purposes. Suitable functional groups that can be used to attachmoieties together include, but are not limited to, amines, anhydrides,hydroxyl groups, carboxy groups, thiols, and the like. An activatingagent, such as a carbodiimide, can be used to form a direct linkage. Awide variety of activating agents are known in the art and are suitablefor conjugating one entity to a second entity.

In other embodiments, entities of a multifunctional agent embraced bythe present invention are indirectly covalently linked to each other viaa linker group. Such a linker group may also be referred to as a linkeror a linking entity. This can be accomplished by using any number ofstable bifunctional agents well known in the art, includinghomofunctional and heterofunctional agents (for examples of such agents,see, e.g., Pierce Catalog and Handbook). The use of a bifunctionallinker differs from the use of an activating agent in that the formerresults in a linking moiety being present in the resulting conjugate(agent), whereas the latter results in a direct coupling between the twomoieties involved in the reaction. The role of a bifunctional linker maybe to allow reaction between two otherwise inert moieties. Alternativelyor additionally, the bifunctional linker that becomes part of thereaction product may be selected such that it confers some degree ofconformational flexibility to the targeting agent in relation to thedetecting moiety (e.g., the bifunctional linker comprises a straightalkyl chain containing several atoms, for example, the straight alkylchain contains between 2 and 10 carbon atoms). Alternatively oradditionally, the bifunctional linker may be selected such that thelinkage formed between a targeting agent and therapeutic agent iscleavable, e.g. hydrolysable (for examples of such linkers, see e.g.U.S. Pat. Nos. 5,773,001; 5,739,116 and 5,877,296, each of which isincorporated herein by reference in its entirety). Such linkers, forexample, may be used when higher activity of certain entities, such as atargeting agent (e.g., compound of formula I) and/or of a therapeuticentity is observed after hydrolysis of the conjugate. Exemplarymechanisms by which an entity may be cleaved from a multifunctionalagent include hydrolysis in the acidic pH of the lysosomes (hydrazones,acetals, and cis-aconitate-like amides), peptide cleavage by lysosomalenzymes (the capthepsins and other lysosomal enzymes), and reduction ofdisulfides). Another mechanism by which such an entity is cleaved fromthe multifunctional agent includes hydrolysis at physiological pH extra-or intra-cellularly. This mechanism applies when the crosslinker used tocouple one entity to another entity is a biodegradable/bioerodiblecomponent, such as polydextran and the like.

For example, hydrazone-containing multifunctional agents can be madewith introduced carbonyl groups that provide the desired releaseproperties. Multifunctional agents can also be made with a linker thatcomprise an alkyl chain with a disulfide group at one end and ahydrazine derivative at the other end. Linkers containing functionalgroups other than hydrazones also have the potential to be cleaved inthe acidic milieu of lysosomes. For example, multifunctional agents canbe made from thiol-reactive linkers that contain a group other than ahydrazone that is cleavable intracellularly, such as esters, amides, andacetals/ketals.

Another example of class of pH sensitive linkers are the cis-aconitates,which have a carboxylic acid group juxtaposed to an amide group. Thecarboxylic acid accelerates amide hydrolysis in the acidic lysosomes.Linkers that achieve a similar type of hydrolysis rate acceleration withseveral other types of structures can also be used.

Another potential release method for targeting agents is the enzymatichydrolysis of peptides by the lysosomal enzymes. In one example, apeptidic toxin is attached via an amide bond to para-aminobenzyl alcoholand then a carbamate or carbonate is made between the benzyl alcohol andthe therapeutic agent. Cleavage of the peptide leads to collapse of theamino benzyl carbamate or carbonate, and release of the therapeuticagent. In another example, a phenol can be cleaved by collapse of thelinker instead of the carbamate. In another variation, disulfidereduction is used to initiate the collapse of a para-mercaptobenzylcarbamate or carbonate.

Useful linkers which may be used as a linking entity of amultifunctional agent provided herein include, without limitation:polyethylene glycol, a copolymer of ethylene glycol, a polypropyleneglycol, a copolymer of propylene glycol, a carboxymethylcellulose, apolyvinyl pyrrolidone, a poly-1,3-dioxolane, a poly-1,3,6-trioxane, anethylene/maleic anhydride copolymer, a polyaminoacid, a dextran n-vinylpyrrolidone, a poly n-vinyl pyrrolidone, a propylene glycol homopolymer,a propylene oxide polymer, an ethylene oxide polymer, a polyoxyethylatedpolyol, a polyvinyl alcohol, a linear or branched glycosylated chain, apolyacetal, a long chain fatty acid, a long chain hydrophobic aliphaticgroup.

Embraced also herein are multifunctional agents that include at leastone entity which involves non-covalent association. Examples ofnon-covalent interactions include, but are not limited to, hydrophobicinteractions, electrostatic interactions, dipole interactions, van derWaals interactions, and hydrogen bonding. Irrespective of the nature ofthe binding, interaction, or coupling, the association between a firstentity and a second entity is, in some embodiments, selective, specificand strong enough so that the second entity contained in the agent doesnot dissociate from the first entity before or during transport/deliveryto and into the target. Thus, Association amongst multiple entities of amultifunctional agent may be achieved using any chemical, biochemical,enzymatic, or genetic coupling known to one skilled in the art.

EXEMPLIFICATION

As depicted in the Examples below, in certain exemplary embodiments,compounds are prepared according to the following general procedures. Itwill be appreciated that, although the general methods depict thesynthesis of certain compounds of the present invention, the followinggeneral methods, and other methods known to one of ordinary skill in theart, can be applied to all compounds and subclasses and species of eachof these compounds, as described herein.

In certain embodiments, compounds of formula I are prepared according tothe procedure outlined in Scheme 1.

The syntheses were executed according to a general scheme starting froma given ketone and reacting it under Strecker reaction conditions, usingsodium cyanide and 4-amino-2-fluoro-N-methylbenzamide. The resultingcyanamine was then reacted with 4-cyano-3-trifluoromethylaniline (X═CH)or a 2-cyano-3-trifluoromethyl-5-aminopyridine (X═N) in the present ofthiophosgene to give the desired thiohydantoins after acid hydrolysis ofthe intermediate imine.

Example 1

Strecker Reaction

A general procedure for the first step of the synthesis of compounds offormula I follows. To a mixture of 4-amino-2-fluoro-N-methylbenzamide(0.3 mmol) and desired ketone (1.0-2.0 eq) in glacial acetic acid (2 mL)was added NaCN (100 mg, 2.0 mmol, 7.0 eq), and the mixture was heated to80° C. overnight. The solvent was then removed under reduced pressureand the residue was dissolved in water (20 mL), then pH was brought toneutrality with aqueous saturated NaHCO₃ solution. The mixture wasextracted with ethyl acetate (3×50 mL), and the combined organic layerswere dried over anhydrous Na₂SO₄. The solution was filtered andconcentrated under reduced pressure and the resulting residue waschromatographed on a short path silica gel column using the gradienthexane/ethyl acetate 2/1 to 1/1.5 (v/v) to yield each desired product inmore than 85% yield.

Example 2

Thiohydantoin Synthesis

A general procedure for the second step of the synthesis of compounds offormula I follows. Thiophosgene (5.1 uL, 66 umol) was added dropwise toa solution of 5-amino-2-cyano-3-trifluoromethylpyridine or4-amino-2-(trifluoromethyl)benzonitrile (60 umol) and the given Streckerproducts above N-methyl-4-(1-cyanocycloalkylamino)-2-fluorobenzamides(60 umol) in dry DMA (0.6 mL) under argon at 0° C. After 5 min, thesolution was stirred overnight at 60 C. At room temperature, thismixture was then diluted with MeOH (1 mL) and aq. 2.0 N HCl (0.5 mL),and the reaction was brought to reflux for 2 hours. After cooling toambient temperature, the reaction mixture was poured into ice water (10mL) and extracted with EtOAc (3×20 mL). The combined organic layers werebriefly dried over anhydrous Mg₂SO₄, concentrated and the resultingresidue was chromatographed on silica gel using the gradient systemhexane/ethyl acetate 2/1 to 1.5/1 (v/v) to yield the desiredthiohydantoin in yields up to 90%.

The following non-limiting examples illustrate analytical data obtainedfor compounds of formula I produced by the synthesis of Scheme 1. Wherea given compound can exist as two or more enantiomers or diastereomers,such isomers can be separated by techniques well known in the art suchas HPLC. By way of example, preparative HPLC purifications can becarried out utilizing a Shimadzu [Prominence LC-20AP], equipped with aChiralpak AGP column (50×21.2 mm, 50 utilizing the following method:Solvent A=Acetonitrile, Solvent B=Water; Gradient=95% solvent B to 10%solvent B over 20 min with a flow rate of 10 mL/min. Analytical LCMSdata can be acquired using a Shimadzu [LCMS-2020] equipped with aSHIMPAK, XR ODS-II column (50×2 mm) utilizing the following method: FlowRate=0.2 mL/min, Solvent A=Acetonitrile, Solvent B=0.1% TFA in water;Gradient=Initial 95% of solvent B to 10% solvent B over 10 min followedby 10% solvent B for an additional 10 min.

Analytical data for compound I-8:4-(3-(4-cyano-3-(trifluoromethyl)phenyl)-4-oxo-2-thioxo-1,3-diazaspiro[4.4]nonan-1-yl)-2-fluoro-N-methylbenzamide.

This compound was isolated as an off-white foam. ¹HNMR (CDCl3): 8.28 (t,1 H, J=8.5 Hz), 7.79 (d, 1 H, J=8.3 Hz), 7.96 (bs, 1 H), 7.84 (dd, 1 H,J=8.3 Hz, J=1.5 Hz), 7.27 (dd, 1 H, J=8.3 Hz, J=1.8 Hz), 7.17 (dd, 1 H,J=11.7 Hz, J=1.5 Hz), 6.71 (m, 1 H), 3.07 (d, 3 H, J=4.7 Hz), 2.36 (m, 2H), 2.16 (m, 2 H), 1.91 (m, 2 H), 1.56 (m, 2 H). ¹⁹FNMR (CDCl₃): −61.98,−110.64. LRMS [M+H]+ found: 491.22; calculated: 491.12.

Analytical data for compound I-9:4-(3-(4-cyano-3-(trifluoromethyl)phenyl)-4-oxo-2-thioxo-1,3-diazaspiro[4.5]decan-1-yl)-2-fluoro-N-methylbenzamide.

The compound was obtained as an off-white foam. ¹HNMR (CDCl₃): 8.27 (t,1 H, J=8.4 Hz), 7.98 (d, 1 H, J=8.3 Hz), 7.93 (bs, 1 H), 7.82 (dd, 1 H,J=8.2 Hz, J=1.6 Hz), 7.19 (dd, 1 H, J=8.3 Hz, J=1.8 Hz), 7.08 (dd, 1 H,J=11.6 Hz, J=1.6 Hz), 6.70 (m, 1 H), 3.08 (d, 3 H, J=4.7 Hz), 2.07 (m, 4H), 1.70 (m, 6 H). ¹⁹FNMR (CDCl₃): −61.97, −110.92. LRMS [M+H]+ found:505.30; calculated: 505.13.

Analytical data for compound I-10:4-(3-(4-cyano-3-(trifluoromethyl)phenyl)-4-oxo-2-thioxo-1,3-diazaspiro[4.6]undecan-1-yl)-2-fluoro-N-methylbenzamide.

This compound was isolated as off-white solid. ¹HNMR (CDCl₃): 8.28 (t, 1H, J=8.4 Hz), 7.98 (d, 1 H, J=8.3 Hz), 7.93 (bs, 1 H), 7.82 (dd, 1 H,J=8.2 Hz, J=1.6 Hz), 7.24 (dd, 1 H, J=8.3 Hz, J=1.6 Hz), 7.14 (dd, 1 H,J=11.6 Hz, J=1.5 Hz), 6.72 (m, 1 H), 3.08 (d, 3 H, J=4.7 Hz), 2.28 (m, 2H), 2.17 (m, 2 H), 1.81 (m, 2 H), 1.60 (m, 2 H), 1.44 (m, 2 H), 1.32 (m,2 H). ¹⁹FNMR (CDCl₃): −61.98, −110.82. LRMS [M+H]+ found: 519.38;calculated: 519.15.

Analytical data for racemic compound I-1:4-(3-(4-cyano-3-(trifluoromethyl)phenyl)-7,7-dimethyl-4-oxo-2-thioxo-1,3-diazaspiro[4.5]decan-1-yl)-2-fluoro-N-methylbenzamide.

This compound was synthesized in 70% overall yield as an off-whitepowder. ¹HNMR (CDCl₃): 8.27 (t, 1 H, J=8.4 Hz), 7.98 (d, 1 H, J=8.3 Hz),7.92 (bs, 1 H), 7.80 (dd, 1 H, J=8.2 Hz, J=1.7 Hz), 7.17 (dd, 1 H, J=8.3Hz, J=1.7 Hz), 7.07 (dd, 1 H, J=11.6 Hz, J=1.6 Hz), 6.70 (m, 1 H), 3.08(d, 3 H, J=4.7 Hz), 2.27 (m, 1 H), 2.17 (m, 1 H), 1.93 (m, 1 H), 1.67(m, 1 H), 1.62 (m, 1 H), 1.57 (m, 1 H), 1.52 (m, 2 H), 1.20 (s, 3 H),0.95 (s, 3 H). ¹⁹FNMR (CDCl₃): −61.98, −110.89. LRMS [M+H]+ found:533.33; calculated: 533.17.

Analytical data for compound I-11:4-(3-(4-cyano-3-(trifluoromethyl)phenyl)-8,8-dimethyl-4-oxo-2-thioxo-1,3-diazaspiro[4.5]decan-1-yl)-2-fluoro-N-methylbenzamide.

This compound was isolated as an off-white powder. ¹HNMR (CDCl₃): 8.30(t, 1 H, J=8.4 Hz), 7.98 (d, 1 H, J=8.3 Hz), 7.93 (bs, 1 H), 7.82 (dd, 1H, J=8.2 Hz, J=1.6 Hz), 7.22 (dd, 1 H, J=8.3 Hz, J=1.6 Hz), 7.11 (dd, 1H, J=11.6 Hz, J=1.5 Hz), 6.72 (m, 1 H), 3.08 (d, 3 H, J=4.7 Hz), 2.04(m, 2 H), 1.93 (m, 4 H), 1.37 (m, 2 H), 0.99 (s, 3 H), 0.73 (s, 3 H).¹⁹FNMR (CDCl₃): −61.98, −110.75. LRMS [M+H]+ found: 533.33; calculated:533.17.

Analytical data for compound I-5:4-(3-(4-cyano-3-(trifluoromethyl)phenyl)-7,7,9,9-tetramethyl-4-oxo-2-thioxo-1,3-diazaspiro[4.5]decan-1-yl)-2-fluoro-N-methylbenzamide.

This compound was isolated as a beige foam. ¹HNMR (CDCl₃): 8.21 (t, 1 H,J=8.4 Hz), 7.90 (d, 1 H, J=8.3 Hz), 7.85 (bs, 1 H), 7.73 (dd, 1 H, J=8.2Hz, J=1.2 Hz), 7.12 (dd, 1 H, J=8.3 Hz, J=1.2 Hz), 7.02 (dd, 1 H, J=11.6Hz, J=1.2 Hz), 6.64 (m, 1 H), 3.01 (d, 3 H, J=4.7 Hz), 1.94 (d, 2H,J=14.4 Hz), 1.62 (d, 2H, J=14.4 Hz), 1.50 (s, 2 H), 1.17 (s, 6 H), 0.83(s, 6 H). ¹⁹FNMR (CDCl₃): −61.98, −110.89. LRMS [M+H]+ found: 561.29;calculated: 561.20.

Analytical data for racemic compound I-3:4-(3-(6-cyano-5-(trifluoromethyl)pyridin-3-yl)-7,7-dimethyl-4-oxo-2-thioxo-1,3-diazaspiro[4.5]decan-1-yl)-2-fluoro-N-methylbenzamide.

This compound was isolated as an off-white foam. ¹HNMR (CDCl₃): 9.06 (d,1 H, J=1.9 Hz), 8.33 (d, 1 H, J=1.9 Hz), 8.29 (t, 1 H, J=8.4 Hz), 7.18(dd, 1 H, J=8.4 Hz, J=1.6 Hz), 7.07 (dd, 1 H, J=11.5 Hz, J=1.5 Hz), 6.71(m, 1 H), 3.08 (d, 3 H, J=4.7 Hz), 2.30 (m, 1 H), 2.18 (m, 1 H), 1.94(m, 1 H), 1.72 (m, 1 H), 1.63 (m, 1 H), 1.57 (m, 1 H), 1.52 (m, 2 H),1.20 (s, 3 H), 0.94 (s, 3 H). ¹⁹FNMR (CDCl₃): −61.87, −110.71. LRMS[M+H]+ found: 534.31; calculated: 534.16.

Analytical data for compound I-26:4-(3-(4-cyano-3-(trifluoromethyl)phenyl)-4-oxo-2-thioxoimidazolidin-1-yl)-2-fluoro-N-methylbenzamide.

This compound was isolated as a white to off-white powder. ¹HNMR(CDCl₃): 8.26 (t, 1 H, J=8.4 Hz), 8.02 (d, 1 H, J=8.3 Hz), 7.91 (bs, 1H), 7.79 (m, 2 H), 7.45 (dd, 1 H, J=10.7 Hz, J=1.3 Hz), 6.71 (m, 1 H),4.71 (s, 2 H), 3.06 (d, 3 H, J=4.7 Hz). ¹⁹FNMR (CDCl₃): −62.05, −110.31.LRMS [M+H]+ found: 437.19; calculated: 437.07.

Analytical data for compound (+)-I-12:4-(1-(4-cyano-3-(trifluoromethyl)phenyl)-5-oxo-2-thioxooctahydro-1′H-spiro[imidazolidine-4,2′-naphthalen]-3-yl)-2-fluoro-N-methylbenzamide.

This compound was isolated as off-white foam. ¹HNMR (CDCl₃): 8.30 (t, 1H, J=8.4 Hz), 7.98 (d, 1 H, J=8.3 Hz), 7.93 (bs, 1 H), 7.82 (dd, 1 H,J=8.2 Hz, J=1.8 Hz), 7.21 (dd, 1 H, J=8.3 Hz, J=1.7 Hz), 7.11 (dd, 1 H,J=11.6 Hz, J=1.6 Hz), 6.73 (m, 1 H), 3.08 (d, 3 H, J=4.7 Hz), 2.62 (m, 1H), 2.35 (m, 1 H), 2.10-1.09 (m, 14 H). ¹⁹FNMR (CDCl₃): −61.96, −110.72.LRMS [M+H]+ found: 559.32; calculated: 559.18.

Analytical data for compound I-13:4-(3-(6-cyano-5-(trifluoromethyl)pyridin-3-yl)-4-oxo-2-thioxo-1,3-diazaspiro[4.4]nonan-1-yl)-2-fluoro-N-methylbenzamide.

This compound was isolated as off-white foam. ¹HNMR (CDCl₃): 9.09 (d, 1H, J=2.0 Hz), 8.35 (d, 1 H, J=1.9 Hz), 8.30 (t, 1 H, J=8.4 Hz), 7.27(dd, 1 H, J=8.4 Hz, J=1.8 Hz), 7.17 (dd, 1 H, J=11.6 Hz, J=1.7 Hz), 6.71(m, 1 H), 3.08 (d, 3 H, J=4.7 Hz), 2.37 (m, 2 H), 2.19 (m, 2 H), 1.92(m, 2 H), 1.57 (m, 2 H). ¹⁹FNMR (CDCl₃): −61.87, −110.47. LRMS [M+H]+found: 492.27; calculated: 492.11.

Analytical data for compound I-14:4-(3-(6-cyano-5-(trifluoromethyl)pyridin-3-yl)-4-oxo-2-thioxo-1,3-diazaspiro[4.5]decan-1-yl)-2-fluoro-N-methylbenzamide.

This compound was obtained as off-white foam. ¹HNMR (CDCl₃): 9.07 (d, 1H, J=1.7 Hz), 8.33 (d, 1 H, J=1.7 Hz), 8.29 (t, 1 H, J=8.4 Hz), 7.19(dd, 1 H, J=8.3 Hz, J=1.4 Hz), 7.17 (dd, 1 H, J=11.5 Hz, J=1.4 Hz), 6.70(m, 1 H), 3.08 (d, 3 H, J=4.7 Hz), 2.09 (m, 4 H), 1.70 (m, 6 H). ¹⁹FNMR(CDCl₃): −61.87, −110.75. LRMS [M+H]+ found: 506.28; calculated: 506.13.

Analytical data for compound I-15:4-(3-(6-cyano-5-(trifluoromethyl)pyridin-3-yl)-4-oxo-2-thioxo-1,3-diazaspiro[4.6]undecan-1-yl)-2-fluoro-N-methylbenzamide.

This compound was isolated as off-white foam. ¹HNMR (CDCl₃): 9.06 (d, 1H, J=1.9 Hz), 8.33 (d, 1 H, J=1.9 Hz), 8.29 (t, 1 H, J=8.4 Hz), 7.24(dd, 1 H, J=8.4 Hz, J=1.6 Hz), 7.13 (dd, 1 H, J=11.5 Hz, J=1.5 Hz), 6.71(m, 1 H), 3.08 (d, 3 H, J=4.7 Hz), 2.30 (m, 2 H), 2.18 (m, 2 H), 1.82(m, 2 H), 1.61 (m, 2 H), 1.45 (m, 2 H), 1.32 (m, 2 H). ¹⁹FNMR (CDCl₃):−61.87, −110.47. LRMS [M+H]+ found: 520.30; calculated: 520.15.

Analytical data for compound I-16:4-(3-(6-cyano-5-(trifluoromethyl)pyridin-3-yl)-8,8-dimethyl-4-oxo-2-thioxo-1,3-diazaspiro[4.5]decan-1-yl)-2-fluoro-N-methylbenzamide.

¹HNMR (CDCl3): 9.06 (d, 1H, J=1.7 Hz), 8.33 (d, 1H, J=1.7 Hz), 8.30 (t,1 H, J=8.4 Hz), 7.22 (dd, 1 H, J=8.3 Hz, J=1.6 Hz), 7.11 (dd, 1 H,J=11.6 Hz, J=1.5 Hz), 6.72 (m, 1 H), 3.08 (d, 3 H, J=4.7 Hz), 2.04-1.94(m, 6H), 1.37 (m, 2 H), 0.99 (s, 3 H), 0.73 (s, 3 H). ¹⁹FNMR (CDCl₃):−61.87, −110.57. LRMS [M+H]+ found: 534.31; calculated: 534.15.

Analytical data for compound (+)-I-17:4-(1-(6-cyano-5-(trifluoromethyl)pyridin-3-yl)-5-oxo-2-thioxooctahydro-1′H-spiro[imidazolidine-4,2′-naphthalen]-3-yl)-2-fluoro-N-methylbenzamide.

This compound was obtained as off-white foam. ¹HNMR (CDCl₃): 9.06 (d, 1H, J=1.9 Hz), 8.32 (d, 1 H, J=1.9 Hz), 8.30 (t, 1 H, J=8.4 Hz), 7.21(dd, 1 H, J=8.4 Hz, J=1.7 Hz), 7.11 (dd, 1 H, J=11.5 Hz, J=1.7 Hz), 6.72(m, 1 H), 3.09 (d, 3 H, J=4.7 Hz), 2.61 (m, 1 H), 2.34 (m, 1 H),2.10-1.09 (m, 14 H). ¹⁹FNMR (CDCl₃): −61.86, −110.54. LRMS [M+H]+ found:560.31; calculated: 560.18.

Analytical data for compound I-27:4-(3-(6-cyano-5-(trifluoromethyl)pyridin-3-yl)-4-oxo-2-thioxoimidazolidin-1-yl)-2-fluoro-N-methylbenzamide.

¹HNMR (CDCl₃): 9.03 (d, 1H, J=1.7 Hz), 8.28 (d, 1H, J=1.7 Hz), 8.25 (t,1 H, J=8.4 Hz), 7.79 (dd, 1 H, J=8.4 Hz, J=1.6 Hz), 7.45 (dd, 1 H,J=10.7 Hz, J=1.3 Hz), 6.71 (m, 1 H), 4.75 (s, 1 H), 4.15 (d, 1H, J=6.7Hz), 3.06 (d, 3 H, J=4.7 Hz). ¹⁹FNMR (CDCl₃): −61.97, −110.14. LRMS[M+H]+ found: 438.18; calculated: 438.06.

Analytical Data for Compound (+)-I-6:

¹HNMR (CDCl₃): 9.06 (d, 1H, J=1.7 Hz), 8.33 (d, 1H, J=1.7 Hz), 8.28 (t,1 H, J=8.4 Hz), 7.11 (dd, 1 H, J=8.3 Hz, J=1.6 Hz), 7.00 (dd, 1 H,J=11.6 Hz, J=1.5 Hz), 6.72 (m, 1 H), 3.01 (d, 3 H, J=4.7 Hz), 2.16-2.03(m, 3H), 1.77 (m, 2 H), 1.61-1.55 (m, 8 H), 1.40-1.29 (m, 4 H). ¹⁹FNMR(CDCl₃): −61.88, −110.70. LRMS [M+H]+ found: 574.26; calculated: 574.6.

Analytical data for compound (+)-I-18:4-((7R)-3-(4-cyano-3-(trifluoromethyl)phenyl)-7-methyl-4-oxo-2-thioxo-1,3-diazaspiro[4.4]nonan-1-yl)-2-fluoro-N-methylbenzamide.

¹HNMR (CDCl₃): 8.23 (m, 1H), 7.90 (d, 1H, J=8.2 Hz), 7.75 (d, 1 H, J=8.2Hz), 7.21 (m, 1H), 7.10 (m, 1 H), 6.72 (m, 1 H), 3.08 (d, 3 H, J=4.7Hz), 2.45 (m, 3H), 2.36 (m, 1H), 2.28 (m, 1H), 2.04 (m, 1H), 1.58 (m,1H), 1.45 (m 1H), 0.96 (d, 1H, J=6.1 Hz), 0.85 (d, 1H, J=6.1 Hz). ¹⁹FNMR(CDCl₃): −61.98, −110.59. LRMS [M+H]+ found: 505.30; calculated: 505.50.

Analytical data for compound (+)-I-19:4-(3-(4-cyano-3-(trifluoromethyl)phenyl)-7-methyl-4-oxo-2-thioxo-1,3-diazaspiro[4.5]decan-1-yl)-2-fluoro-N-methylbenzamide.

¹HNMR (CDCl₃): 8.27 (t, 1 H, J=8.4 Hz), 7.97 (d, 1 H, J=8.3 Hz), 7.93(d, 1 H, J=1.5 Hz), 7.82 (d, 1 H, J=1.5 Hz), 7.19 (d, 1 H, J=8.3 Hz),7.09 (d, 1H, J=10 Hz), 6.73 (m, 1 H), 3.08 (d, 3 H, J=4.5 Hz), 2.6 (m,1H), 2.11-2.04 (m, 2H), 1.77 (m, 2H), 1.63 (m, 2H), 1.26 (m, 2 H), 0.92(d, 3H, 18.4 Hz). ¹⁹FNMR (CDCl₃): −61.96, −110.89. LRMS [M+H]+ found:519.25; calculated: 519.53.

Analytical data for compound (+)-I-20:4-((7R)-3-(4-cyano-3-(trifluoromethyl)phenyl)-7-methyl-4-oxo-2-thioxo-1,3-diazaspiro[4.5]decan-1-yl)-2-fluoro-N-methylbenzamide.

¹HNMR (CDCl₃): 8.27 (t, 1 H, J=8.4 Hz), 7.97 (d, 1 H, J=8.3 Hz), 7.92(d, 1 H, J=1.4), 7.82 (d, 1 H, J=6.5 Hz), 7.19 (d, 1 H, J=8.3 Hz), 7.09(d, 1H, J=10 Hz), 6.73 (m, 1 H), 3.08 (d, 3 H, J=4.5 Hz), 2.6 (m, 1H),2.11-2.04 (m, 2H), 1.77 (m, 2H), 1.63 (m, 2H), 1.26 (m, 2H), 0.92 (d,3H, 18.4 Hz). ¹⁹FNMR (CDCl₃): −61.96, −110.89. LRMS [M+H]+ found:519.25; calculated: 519.53.

Analytical data for compound (+)-I-21:4-(1′-(4-cyano-3-(trifluoromethyl)phenyl)-5′-oxo-2′-thioxospiro[bicyclo[3.2.0]hept[2]ene-6,4′-imidazolidin]-3′-yl)-2-fluoro-N-methylbenzamide.

¹HNMR (CDCl₃): 8.28 (t, 1 H, J=8.4 Hz), 7.90 (d, 1 H, J=8.4 Hz), 7.85(d, 1 H, J=1.4), 7.72 (m, 1 H), 7.31 (d, 1 H, J=8.4 Hz), 7.22 (d, 1H,J=10 Hz), 6.66 (m, 1 H), 5.85 (m, 1H), 5.66 (m, 1H), 3.41 (m, 1H), 3.01(d, 3 H, J=4.5 Hz), 2.94 (m, 1H), 2.76 (m, 1H), 2.58 (m, 1H), 2.53 (m,2H). ¹⁹FNMR (CDCl₃): −62.00, −110.53. LRMS [M+H]+ found: 515.30;calculated: 515.49.

Analytical data for compound (+)-I-22:4-((7R)-3-(6-cyano-5-(trifluoromethyl)pyridin-3-yl)-7-methyl-4-oxo-2-thioxo-1,3-diazaspiro[4.4]nonan-1-yl)-2-fluoro-N-methylbenzamide.

¹HNMR (CDCl₃): 9.08 (d, 1H, J=2.0 Hz), 8.34 (d, 1H, J=2.0 Hz), 8.31 (m,1H), 7.28 (m, 1H), 7.17 (m, 1H), 6.71 (m, 1H), 3.08 (d, 3 H, J=4.7 Hz),2.45 (m, 3H), 2.36 (m, 1H), 2.28 (m, 1H), 2.04 (m, 1H), 1.67 (m, 1H),1.54 (m 1H), 1.04 (d, 1H, J=6.4 Hz), 0.93 (d, 1H, J=6.4 Hz). ¹⁹FNMR(CDCl₃): −61.87, −110.47. LRMS [M+H]+ found: 506.28; calculated: 506.49.

Analytical data for compound (+)-I-23:4-(3-(6-cyano-5-(trifluoromethyl)pyridin-3-yl)-7-methyl-4-oxo-2-thioxo-1,3-diazaspiro[4.5]decan-1-yl)-2-fluoro-N-methylbenzamide.

¹HNMR (CDCl₃): 9.07 (d, 1H, J=2.0 Hz), 8.33 (d, 1H, J=2.0 Hz), 8.28 (t,1H, J=8.4 Hz), 7.26 (d, 1H, J=3.4 Hz), 7.08 (d, 1H, J=1.7 Hz), 6.71 (m,1H), 4.25 (bs, 1H), 3.08 (d, 3H, J=4.5 Hz), 2.6 (m, 1H), 2.11-2.04 (m,2H), 1.77 (m, 2H), 1.63 (m, 2H), 1.26 (m, 2 H), 0.92 (d, 3H, 18.4 Hz).¹⁹FNMR (CDCl₃): −61.86, −110.70. LRMS [M+H]+ found: 520.23; calculated:520.51.

Analytical data for compound (+)-I-24:4-((7R)-3-(6-cyano-5-(trifluoromethyl)pyridin-3-yl)-7-methyl-4-oxo-2-thioxo-1,3-diazaspiro[4.5]decan-1-yl)-2-fluoro-N-methylbenzamide.

¹HNMR (CDCl₃): 9.08 (d, 1H, J=2.0 Hz), 8.33 (d, 1H, J=2.0 Hz), 8.28 (t,1H, J=8.4 Hz), 7.26 (d, 1H, J=3.4 Hz), 7.08 (d, 1H, J=1.7 Hz), 6.71 (m,1H), 4.25 (bs, 1H), 3.08 (d, 3H, J=4.5 Hz), 2.6 (m, 1H), 2.11-2.04 (m,2H), 1.77 (m, 2H), 1.63 (m, 2H), 1.26 (m, 2 H), 0.92 (d, 3H, 18.4 Hz).¹⁹FNMR (CDCl₃): −61.86, −110.71. LRMS [M+H]+ found: 520.23; calculated:520.51.

Analytical data for compound (+)-I-25:4-(1′-(6-cyano-5-(trifluoromethyl)pyridin-3-yl)-5′-oxo-2′-thioxospiro[bicyclo[3.2.0]hept[2]ene-6,4′-imidazolidin]-3′-yl)-2-fluoro-N-methylbenzamide.

¹HNMR (CDCl₃): 9.08 (d, 1H, J=2.1 Hz), 8.33 (t, 1H, J=8.4 Hz), 8.31 (d,1H, J=2.1 Hz), 7.38 (d, 1H, J=1.8 Hz), 7.29 (d, 1 H, J=9.8 Hz), 6.71 (m,1H), 5.92 (m, 1 H), 5.74 (m, 1H), 3.50 (m, 1H), 3.08 (d, 3 H, J=4.7 Hz),3.00 (m, 1H), 2.8 (m, 1H), 2.66-2.59 (m, 3 H). ¹⁹FNMR (CDCl₃): −61.89,−110.34. LRMS [M+H]+ found: 516.29; calculated: 516.51.

Analytical data for compound I-7:4-(3-(6-cyano-5-(trifluoromethyl)pyridin-3-yl)-7,7,9,9-tetramethyl-4-oxo-2-thioxo-1,3-diazaspiro[4.5]decan-1-yl)-2-fluoro-N-methylbenzamide.

This compound was isolated as a beige foam. ¹HNMR (CDCl₃): 8.9 (d, 1H,J=1.5 Hz), 8.24 (d, 1H, J=1.5 Hz), 8.22 (t, 1 H, J=8.4 Hz), 7.19 (dd, 1H, J=8.3 Hz, J=1.6 Hz), 7.03 (dd, 1 H, J=11.6 Hz, J=1.5 Hz), 6.65 (m, 1H), 3.01 (d, 3 H, J=4.7 Hz), 1.97 (d, 2H, J=14.4 Hz), 1.62 (d, 2H,J=14.4 Hz), 1.50 (s, 2 H), 1.17 (s, 6H), 0.83 (s, 6H). ¹⁹FNMR (CDCl₃):−61.89, −110.57. LRMS [M+H]+ found: 562.28; calculated: 562.59.

Additional compounds of formula I were prepared in a mannersubstantially similar to that described above.

Example 17

In Vitro GFP Reporter Assay

Compounds of the present invention were assayed in an in vitro GFP(green fluorescent protein) reporter assay, which can be used todetermine the effect of compounds on AR transcriptional activity. LNCaP(prostate cancer) cells were engineered to express an AR-regulated GFPreporter (Pb.PSE.EGFP), such that GFP expression indicates ARtranscriptional activity. A representative procedure for this assayfollows.

LNCaP-Pb.PSE.EGFP cells overexpressing either wild-type AR (WT) or theF876L mutant AR (FL) were treated with vehicle (DMSO) or with either 1uM or 10 uM of Enzalutamide or ARN509, or 10 uM of a compound of formulaI. After 4 days the treated cells were subjected to flow cytometricanalysis and the histogram overlays of GFP expression (FL1) weredisplayed Results of certain compounds are depicted (together with thegeometric mean fluorescence intensity is indicated in the legends belowthe histogram) in each of FIGS. 1 through 15. In each of the figures,results for the assay using WT AR is on the left side graph and theF876L mutant AR is on the right side graph. FIG. 16 depicts tabulatedresults obtained for some of the test compounds. In some embodiments, aprovided compound is considered to be a modulator of a tested AR if itresults in similar effects on induced GFP expression as compared with areference androgen or anti-androgen.

Example 18

AR Luciferase Reporter Assay

Compounds of the present invention were assayed in an in vitro ARluciferase reporter assay reporter assay, which can be used to determinethe effect of compounds on AR transcriptional activity. CV1 cells wereengineered to express an AR-regulated luciferase reporter, such thatluciferase expression indicates AR transcriptional activity. Arepresentative procedure for this assay follows.

CV1 cells (10⁶ cells/10 cm plate) were cotransfected with 50 ng of SV40Renilla Luciferase, 5 ug of ARE(4X)-Luciferase, and 10 ug of one pWZL-ARexpression construct using Lipofectamine 2000 (Invitrogen). Transfectionmedia was removed 4-6 hours later and replaced with phenol red freeDME-HG containing 10% charcoal stripped serum. The following day eachplate was split into 24- or 48-well plates, in 10% CSS media, containingthe indicated drugs in triplicate. Twenty-four to forty-eight hourslater, luciferase activity was assayed using Dual-Luciferase ReporterAssay System (Promega) on a 96-well luminometer (Turner Biosystems). Insome embodiments, a provided compound is considered to be a modulator ofa tested AR if it results in similar effects on induced expression ofluciferase in the luciferase reporter assay as compared with a referenceandrogen or anti-androgen.

Example 19

In Vitro Cell Viability Assay

Compounds of the present invention were assayed in an in vitro cellviability assay, which can be used to determine the effect of compoundson LNCaP cell growth and survival. A representative procedure for thisassay follows.

LNCaP-Pb.PSE-EGFP cells for flow cytometric analysis were treated withtest compounds (1 uM or 10 uM) for 4-6 days, changing media and drugevery 2-3 days. Cells were collected using Accumax dissociation solution(Innovative Cell Technologies) and dead cells were counterstained usingTO-PRO3-Iodide (Invitrogen). EGFP expression was measured using theBD-FACSCalibur flow cytometer using the 488 nm laser and 530/30 bandpassfilter to detect EGFP expression, and the 633 nm laser and 661/16bandpass filter to detect TO-PRO3-Iodide labeled dead cells. For eachsample, 2-5×104 cell events were collected and analysis was done usingFlowJo software. FACS-sorting of LNCaP-Pb.PSE.EGFP cells was performedon a BD FACSVantage cell sorter. EGFP expression was detected using the488 nm laser and 530/30 bandpass filter, and DAPI-labeled dead cellswere detected using the 355 nm laser and 450/50 bandpass filter. Resultsof an in vitro cell viability assay are depicted in FIG. 17. In someembodiments, a provided compound is considered to be a modulator of atested AR if it results in similar effects on cell viability as comparedwith a reference androgen or anti-androgen.

Example 20

Compounds of the present invention were also assayed in an in vitro cellviability assay in CWR22PC cancer cells. A representative procedurefollows. CWR22PC cells ectopically expressing wild-type AR or AR F876L,cultured in full-serum containing media, were treated with vehicle(DMSO) or 10 uM of enzalutamide or compound I-1. CellTiterGLO assay wasperformed on days 1, 4, and 7 to determine cell viability. Results of acell viability assay in CWR22PC cells are depicted in FIG. 18. On they-axis RLU means relative light units. More light scattered means moreviable cells. In some embodiments, a provided compound is considered tobe a modulator of a tested AR if it results in similar effects on cellviability as compared with a reference androgen or anti-androgen.

Example 21

Initial Models of AR-Antiandrogen Complex Structures

No structures have been solved experimentally for enzalutamide orARN-509 in complex with AR (agonist or antagonist conformation).Therefore, 3D structures of antiandrogens were first built using thecomputer program Gaussview (version 4.1.2, part of the computer programGaussian 03) and then geometrically optimized in a quantum mechanicalforce field at the level of restricted Hartree-Fock (RHF) 6-31g* usingthe program Gaussian 03. The partial atomic charges were derived fromthe optimized structures by Restrained ElectroStatic Potential (RESP)fitting to the RHF/6-31g* potentials. The other parameters modeling theantiandrogens were taken from the CHARMm22 force field after assigningCHARMm22 atom types to antiandrogens with an in-house program.

The initial AR-antiandrogen complex structures were then modeled withthe molecular modeling program CHARMM5,6. Starting with the atomiccoordinates of AR WT and A ring of 51 in the template crystal structure(PDB accession code, 2AXA), the side chain of residue 761 were replacedwith CHARMm22-parameterized side chain of a leucine in cases of AR F876Land a CH group on the A ring was replaced with a nitrogen in cases ofARN-509. The rest of each antiandrogen was “grown” from the A ring usingthe ideal, unbound structures solved by geometry optimization. Missingside chain atoms were built using standard CHARMm22 parameters andhydrogen atoms were added with the HBUILD module of CHARMM. All thesenewly-introduced atoms without 3D crystal coordinates treated flexibleand the rest under harmonic constraints with the force constant of 100Kcal/mol/Å², each AR-antiandrogen complex structure was energeticallyminimized with 1 round of 100-step steepest decent followed by 2 roundsof 100-step Adopted-Basis Newton-Raphson (ABNR) energy minimization.Harmonic constraints were reset at the beginning of each round ofminimization. No nonbonded cutoff was used. Solvent effects wereimplicitly modeled in this stage with a distance-dependent dielectricconstant.

Example 22

Molecular Dynamics Simulations

The all-atom MD simulations were performed with explicit solvent atomsusing the program CHARMM (version 36a1). Each initial AR-antiandrogenmodel was first centered and overlaid with a 50 Å×50 Å×50 Å cube ofapproximately 47,000 equilibrated water molecules. Any water moleculewhose oxygen atom was within 2.8 Å away from any non-hydrogen atom of ARor antiandrogen was removed. Proper amount of sodium and chloride ionswere automatically added to achieve overall charge neutrality andphysiological level of ion concentration (0.145 M). Their positions wereoptimized with 10 independent trajectories of randomly replacing watermolecules and performing 50 steps of steepest decent and 125 steps ofABNR energy minimization.

The molecular system including AR, antiandrogen, waters, and ions washeated to 298 K and equilibrated with two rounds of 0.1-ns MDsimulations under successively weaker harmonic constraints on AR orantiandrogen atoms. After the MD equilibration, three sets of randomvelocities were assigned to initiate three independent 10-ns MDproductions. The MD equilibration and production were performed usingthe crystal form of rhombic dodecahedron (RHDO) and the canonicalensemble (NVT). A nonbonded cutoff of 10 Å, periodic boundary conditionsin conjunction with Ewald summation method, the leapfrog Verletintegrator, and the Hoover thermostat for pressure and temperature wereused. The timestep was set as 2 fs. Parallel jobs for MD simulationswere run on a computer cluster of Intel Xeon X5650 series (2.66 GHz and4 GB memory for each CPU).

Structural models were visualized in a molecular graphics program, UCSFChimera. The default option used when aligning structures. Results ofthe molecular modeling experiments are depicted in FIGS. 19 through 23.In some embodiments, the AR modulator, agonist or antagonist, asdescribed herein, is one dimensioned to fit within the pocket defined bythe computational model described above.

Example 23

Ligand Binding Assay

The relative binding affinity of DHT and AR antagonists in LNCaP cellsectopically expressing AR WT or AR F876L was determined using acompetition assay in which increasing concentrations of cold competitorare added to cells pre-incubated with 18F-FDHT. The cells werepropagated in RPMI media supplemented with 10% CSS (charcoal-stripped,dextran-treated fetal bovine serum). Cells were trypsinized, washed inPBS, and triplicate cell samples were mixed with 20,000 cpm 18F-FDHT andincreasing amounts of cold competitor (0.1 nM to 10 uM). The solutionswere shaken on an orbital shaker at ambient temperature, and after 1hour the cells were isolated and washed with ice-cold Tris-bufferedsaline using a Brandel cell harvester (Gaithersburg, Md.). All theisolated cell samples were counted using a scintillation counter, withappropriate standards of total activity and blank controls, and thespecific uptake of ¹⁸F-FDHT determined. These data were plotted againstthe concentration of the cold competitor to give sigmoidal displacementcurves. The IC₅₀ values were determined using a one site model and aleast squares curve fitting routine (Origin, OriginLab, Northampton,Mass.) with the R² of the curve fit being >0.99.

These data show that DHT binds to both AR WT and AR F876L expressingcells at a comparable IC50. However, MDV3100 is able to displace¹⁸F-FDHT from the mutant AR F876L cells at a lower IC50 than for AR WTcells, suggesting a higher binding affinity of MDV3100 to the AR F876Lmutant. These data are in line with previous data that showed the mutantAR T877A, which confers agonism on the antiandrogen hydroxyflutamide,binds to hydroxyflutamide (and several other hormones) with a higheraffinity than to AR WT. (Ozers, et al. “The androgen receptor T877Amutant recruits LXXLL and FXXLF peptides differently than wild-typeandrogen receptor in a time-resolved fluorescence resonance energytransfer assay.” Biochemistry (2007) 46, 683).

In some embodiments, a provided compound is considered to be amodulator, agonist or antagonist of a tested AR if it displaces ¹⁸F-FDHTfrom an AR.

While we have described a number of embodiments of this invention, it isapparent that our basic examples may be altered to provide otherembodiments that utilize the compounds and methods of this invention.Therefore, it will be appreciated that the scope of this invention is tobe defined by the appended claims rather than by the specificembodiments that have been represented by way of example.

We claim:
 1. A compound of formula IV:

or a pharmaceutically acceptable salt thereof, wherein: X is CH or N;each R^(b) is independently a substituted or unsubstituted C₁₋₆aliphatic; and n is 0 to
 4. 2. The compound according to claim 1,wherein the compound is:


3. A pharmaceutical composition comprising a compound according to claim1, or a pharmaceutically acceptable salt thereof, and a pharmaceuticallyacceptable carrier, adjuvant, or vehicle.
 4. The compound of claim 1,wherein the compound is represented by the following structural formula:


5. The compound of claim 1, wherein the compound is represented by thefollowing structural formula: